Recombinant production of mixtures of antibodies

ABSTRACT

The invention provides methods for producing mixtures of antibodies from a single host cell clone, wherein, a nucleic acid sequence encoding a light chain and nucleic acid sequences encoding different heavy chains are expressed in a recombinant host cell. The recombinantly produced antibodies in the mixtures according to the invention suitably comprise identical light chains paired to different heavy chains capable of pairing to the light chain, thereby forming functional antigen-binding domains. Mixtures of the recombinantly produced antibodies are also provided by the invention. Such mixtures can be used in a variety of fields.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional patent application of co-pendingapplication Ser. No. 11/593,279, filed Nov. 6, 2006, now U.S. Pat. No.7,429,486 which is a divisional patent application of patent applicationSer. No. 11/039,767, filed Jan. 18, 2005, now U.S. Pat. No. 7,262,028,issued Aug. 28, 2007, which is a continuation of PCT InternationalPatent Application No. PCT/EP2003/007690, filed on Jul. 15, 2003,designating the United States of America, published, in English, asInternational Publication No. WO 2004/009618 A2 on Jan. 29, 2004, whichitself claims the benefit of PCT International Patent Application No.PCT/EP03/50201, filed May 27, 2003, and European Patent Application No.02077953.4, filed Jul. 18, 2002, and U.S. Provisional Patent ApplicationSer. No. 60/397,066, filed Jul. 18, 2002, the contents of the entiretyof each of which are incorporated by reference.

STATEMENT ACCORDING TO 37 C.F.R. §1.52(e)(5) Sequence Listing Submittedon Compact Disc

Pursuant to 37 C.F.R. §1.52(e)(1)(iii), a compact disc containing anelectronic version of the Sequence Listing has been submittedconcomitant with this application, the contents of which are herebyincorporated by reference. A second compact disc is submitted and is anidentical copy of the first compact disc. The discs are labeled “copy 1”and “copy 2,” respectively, and each disc contains one file entitled“0079WO00ORD.ST25.txt” which is 27 KB and created on Jan. 11, 2005.

TECHNICAL FIELD

The invention relates to the field of biotechnology, and moreparticularly, to the field of medicine and the production of antibodies,and even more particularly, to the production of mixtures of antibodies.

BACKGROUND

The essential function of the immune system is the defense againstinfection. The humoral immune system combats molecules recognized asnon-self, such as pathogens, using immunoglobulins. Theseimmunoglobulins, also called antibodies, are raised specifically againstthe infectious agent, which acts as an antigen, upon first contact(Roitt, Essential Immunology, Blackwell Scientific Publications, fifthedition, 1984; all references cited herein are incorporated in theirentirety by reference). Antibodies are multivalent molecules comprisingheavy (H) chains and light (L) chains joined with interchain disulfidebonds. Several isotypes of antibodies are known, including IgG1, IgG2,IgG3, IgG4, IgA, IgD, IgE, and IgM. An IgG contains two heavy and twolight chains. Each chain contains constant (C) and variable (V) regions,which can be broken down into domains designated C_(H1), C_(H2), C_(H3),V_(H), and C_(L), V_(L) (FIG. 1). Antibody binds to antigen via thevariable region domains contained in the Fab portion and, after binding,can interact with molecules and cells of the immune system through theconstant domains, mostly through the Fc portion.

B-lymphocytes can produce antibodies in response to exposure tobiological substances like bacteria, viruses and their toxic products.Antibodies are generally epitope-specific and bind strongly tosubstances carrying these epitopes. The hybridoma technique (Kohler andMilstein, 1975) makes use of the ability of B-cells to producemonoclonal antibodies to specific antigens and to subsequently producethese monoclonal antibodies by fusing B-cells from mice exposed to theantigen of interest to immortalized murine plasma cells. This technologyresulted in the realization that monoclonal antibodies produced byhybridomas could be used in research, diagnostics and therapies to treatdifferent kinds of diseases like cancer and auto-immune-relateddisorders.

Because antibodies that are produced in mouse hybridomas can inducestrong immune responses in humans, it has been appreciated in the artthat antibodies required for successful treatment of humans needed to beless immunogenic or, preferably, non-immunogenic. For this to be done,murine antibodies were first engineered by replacing the murine constantregions with human constant regions (referred to as chimericantibodies). Subsequently, domains between thecomplementarity-determining regions (CDRs) in the variable domains, theso-called framework regions, were replaced by their human counterparts(referred to as humanized antibodies). The final stage in thishumanization process has been the production of fully human antibodies.

In the art, bispecific antibodies, which have binding specificities fortwo different antigens, have also been described. These are generallyused to target a therapeutic or diagnostic moiety, for instance, T-cell,a cytotoxic trigger molecule, or a chelator that binds a radionuclide,that is recognized by one variable region of the antibody to a cell thatis recognized by the other variable region of the antibody, forinstance, a tumor cell (for bispecific antibodies, see Segal et al.,2001).

One very useful method known in the art to obtain fully human monoclonalantibodies with desirable binding properties, employs phage displaylibraries. This is an in vitro, recombinant DNA-based, approach thatmimics key features of the humoral immune response (for phage displaymethods, see, e.g., C. F. Barbas III et al., Phage Display, A laboratorymanual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,2001). For the construction of phage display libraries, collections ofhuman monoclonal antibody heavy- and light-chain variable region genesare expressed on the surface of bacteriophage particles, usually insingle-chain Fv (scFv) or in Fab format. Large libraries of antibodyfragment-expressing phages typically contain more than 10⁹ antibodyspecificities and may be assembled from the immunoglobulin V regionsexpressed in the B lymphocytes of immunized or non-immunizedindividuals.

Alternatively, phage display libraries may be constructed fromimmunoglobulin variable regions that have been partially assembled orrearranged in vitro to introduce additional antibody diversity in thelibrary (semi-synthetic libraries) (De Kruif et al., 1995b). Forexample, in vitro-assembled variable regions contain stretches ofsynthetically produced, randomized or partially randomized DNA in thoseregions of the molecules that are important for antibody specificity.

The genetic information encoding the antibodies identified by phagedisplay can be used for cloning the antibodies in a desired format, forinstance, IgG, IgA or IgM, to produce the antibody with recombinant DNAmethods (Boel et al., 2000).

An alternative method to provide fully human antibodies uses transgenicmice that comprise genetic material encoding a human immunoglobulinrepertoire (Fishwild et al., 1996; Mendez et al., 1997). Such mice canbe immunized with a target antigen and the resulting immune responsewill produce fully human antibodies. The sequences of these antibodiescan be used in recombinant production methods.

Production of monoclonal antibodies is routinely performed by use ofrecombinant expression of the nucleic acid sequences encoding the H andL chains of antibodies in host cells (see, e.g., EP0120694; EP0314161;EP0481790; U.S. Pat. No. 4,816,567; WO 00/63403, the contents of theentirety of each which are incorporated herein by reference).

To date, many different diseases are being treated with either humanizedor fully human monoclonal antibodies. Products based on monoclonalantibodies that are currently approved for use in humans includeHERCEPTIN™ (trastuzumab, anti-Her2/Neu), REOPRO™ (abciximab,anti-Glycoprotein IIB/IIIA receptor), MYLOTARG™ (gemtuzumab, anti-CD33),RITUXAN™ (Rituximab, anti-CD20), SIMULECT™ (basiliximab, anti-CD25),REMICADE™ (infliximab, anti-TNF), SYNAGIS™ (palivizumab, anti-RSV),ZENAPAX™ (daclizumab, IL2-receptor), and CAMPATH™ (alemtuzumab,anti-CD52). Despite these successes, there is still room for newantibody products and for considerable improvement of existing antibodyproducts.

The use of monoclonal antibodies in cancer treatment has shown thatso-called “antigen-loss tumor variants” can arise, making the treatmentwith the monoclonal antibody less effective. Treatment with the verysuccessful monoclonal antibody RITUXIMAB® (anti-CD20) has, for instance,shown that antigen-loss escape variants can occur, leading to relapse ofthe lymphoma (Massengale et al., 2002). In the art, the potency ofmonoclonal antibodies has been increased by fusing them to toxiccompounds, such as radionuclides, toxins, cytokines, and the like. Eachof these approaches, however, has its limitations, includingtechnological and production problems and/or high toxicity.

Furthermore, it appears that the gain in specificity of monoclonalantibodies compared to traditional undefined polyclonal antibodies,comes at the cost of loss of efficacy. In vivo, antibody responses arepolyclonal in nature, i.e., a mixture of antibodies is produced becausevarious B-cells respond to the antigen, resulting in variousspecificities being present in the polyclonal antibody mixture.Polyclonal antibodies can also be used for therapeutic applications, forinstance, for passive vaccination or for active immunotherapy, andcurrently are usually derived from pooled serum from immunized animalsor from humans who recovered from the disease. The pooled serum ispurified into the proteinaceous or gamma globulin fraction, so namedbecause it contains predominantly IgG molecules.

Polyclonal antibodies that are currently used for treatment includeanti-rhesus polyclonal antibodies, gamma globulin for passiveimmunization, anti-snake venom polyclonal (CroFab), THYMOGLOBULIN™ forallograft rejection, anti-digoxin to neutralize the heart drug digoxin,and anti-rabies polyclonal antibodies. In currently marketed therapeuticantibodies, an example of the higher efficacy of polyclonal antibodiescompared to monoclonal antibodies can be found in the treatment of acutetransplant rejection with anti-T-cell antibodies. The monoclonalantibodies on the market (anti-CD25 BASILIXIMAB®) are less efficaciousthan a rabbit polyclonal antibody against thymocytes (THYMOGLOBULIN™)(press releases dated Mar. 12, Apr. 29, and Aug. 26, 2002, onsangstat.com). The use of pooled human sera, however, potentially bearsthe risk of infections with viruses such as HIV or hepatitis, withtoxins such as lipopolysaccharide, with proteinaceous infectious agentssuch as prions, and with unknown infectious agents. Furthermore, thesupply that is available is limited and insufficient for widespreadhuman treatments. Problems associated with the current application ofpolyclonal antibodies derived from animal sera in the clinic include astrong immune response of the human immune system against such foreignantibodies. Therefore, such polyclonals are not suitable for repeatedtreatment or for treatment of individuals that were injected previouslywith other serum preparations from the same animal species.

The art describes the idea of the generation of animals with a humanimmunoglobulin repertoire, which can subsequently be used forimmunization with an antigen to obtain polyclonal antibodies againstthis antigen from the transgenic animals (WO 01/19394, the entirety ofwhich is incorporated herein by reference). However, many technologicalhurdles still will have to be overcome before such a system is apractical reality in larger animals than mice and it will take years ofdevelopment before such systems can provide the polyclonal antibodies ina safe and consistent manner in sufficient quantities. Moreover,antibodies produced from pooled sera, whether being from human or animalorigin, will always comprise a high amount of unrelated and undesiredspecificities, as only a small percentage of the antibodies present in agiven serum will be directed against the antigen used for immunization.It is, for instance, known that in normal, i.e., non-transgenic,animals, about 1% to 10% of the circulating immunoglobulin fraction isdirected against the antigen used for hyper-immunization; hence, thevast majority of circulating immunoglobulins is not specific.

One approach towards expression of polyclonal antibody libraries hasbeen described (WO 95/20401; U.S. Pat. Nos. 5,789,208 and 6,335,163, thecontents of the entirety of each of which are incorporated herein byreference). A polyconal library of Fab antibody fragments is expressedusing a phage display vector and selected for reactivity towards anantigen. To obtain a sub-library of intact polyconal antibodies, theselected heavy and light chain-variable region gene combinations aretransferred en mass as linked pairs to a eukaryotic-expression vectorthat provides constant region genes. Upon transfection of thissub-library into myeloma cells, stable clones produce monoclonalantibodies that can be mixed to obtain a polyclonal antibody mixture.While in theory it would be possible to obtain polyclonal antibodiesdirectly from a single recombinant production process using this methodby culturing a mixed population of transfected cells, potential problemswould occur concerning the stability of the mixed cell population and,hence, the consistency of the produced polyclonal antibody mixture. Thecontrol of a whole population of different cells in a pharmaceuticallyacceptable large-scale process (i.e., industrial) is a daunting task. Itwould seem that characteristics, such as growth rates of the cells andproduction rates of the antibodies, should remain stable for all of theindividual clones of the non-clonal population in order to keep theratio of antibodies in the polyclonal antibody mixture more or lessconstant.

Thus, while the need for mixtures of antibodies may have been recognizedin the art, no acceptable solutions exist to economically make mixturesof antibodies in a pharmaceutically acceptable way.

SUMMARY OF THE INVENTION

The invention provides means for producing a mixture of antibodies inrecombinant hosts.

In one aspect, the invention provides for a method of producing amixture of antibodies in a recombinant host, the method comprisingexpressing in a recombinant host cell a nucleic acid sequence or nucleicacid sequences encoding at least one light chain and at least threedifferent heavy chains that are capable of pairing with at least onelight chain. A further aspect of the invention is the elimination of theproduction of potentially non-functional light-heavy chain pairing byusing pre-selected combinations of heavy and light chains. It has beenrecognized that phage display libraries built from a single light chainand many different heavy chains can encode antibody fragments with verydistinct binding properties. This feature can be used to find differentantibodies having the same light chain but different heavy chains,against the same target or different targets, wherein a target can be awhole antigen or an epitope thereof. Such different targets may, forinstance, be on the same surface (e.g., cell or tissue). Such antibodyfragments obtained by phage display can be cloned into vectors for thedesired format, e.g., IgG, IgA or IgM, and the nucleic acid sequencesencoding these formats can be used to transfect host cells. In oneapproach, H and L chains can be encoded by different constructs that,upon transfection into a cell wherein they are expressed, give rise tointact Ig molecules. When different H chain constructs are transfectedinto a cell with a single L chain construct, H and L chains will beassembled to form all possible combinations. However, in contrast toapproaches where different light chains are expressed, such as for theproduction of bispecific antibodies, this method will result only infunctional binding regions. It would be particularly useful when thehost, for example, a single cell line, is capable of expressingacceptable levels of recombinant antibodies without the necessity tofirst amplify in the cell the nucleic acid sequences encoding theantibodies. The advantage is that cell lines with only a limited copynumber of the nucleic acids are expected to be genetically more stable,because there will be less recombination between the sequences encodingthe heavy chains, than in cell lines where a multitude of these copiesis present. A cell line suitable for use in the methods according to theinvention is the human cell line PER.C6® (human retina cells thatexpress adenovirus E1A and E1B proteins). Using this method, a mixtureof antibodies with defined specificities can be produced from a singlecell clone in a safe, controlled, and consistent manner.

In certain embodiments, the invention provides a method for producing amixture of antibodies in a recombinant host, the method comprisingexpressing a nucleic acid sequence or nucleic acid sequences encoding atleast one light chain and at least three different heavy chains that arecapable of pairing with at least one light chain in a recombinant hostcell. In certain embodiments, the recombinant host cell comprises anucleic acid sequence encoding a common light chain that is capable ofpairing with at least three different heavy chains, such that theproduced antibodies comprise a common light chain. Those of skill in theart will recognize that “common” also refers to functional equivalentsof the light chain of which the amino acid sequence is not identical.Many variants of the light chain exist wherein mutations (deletions,substitutions, additions) are present that do not materially influencethe formation of functional binding regions.

The invention further provides a composition comprising a mixture ofrecombinantly produced antibodies, wherein at least three differentheavy chain sequences are represented in the mixture. In certainembodiments, the light chains of such mixtures have a common sequence.The mixture of antibodies can be produced by the method according to theinvention. Preferably, the mixture of antibodies is more efficaciousthan the individual antibodies it comprises. More preferably, themixture acts synergistically in a functional assay.

The invention further provides a recombinant host cell for producingmixtures of antibodies and methods for making such host cells.

Independent clones obtained from the transfection of nucleic acidsequences encoding a light chain and more than one heavy chain mayexpress the different antibodies in the mixture at different levels. Itis another aspect of the invention to select a clone using a functionalassay for the most potent mixture of antibodies. The invention,therefore, further provides a method for identifying at least one hostcell clone that produces a mixture of antibodies, wherein the mixture ofantibodies has a desired effect according to a functional assay, themethod comprising: (i) providing a host cell with nucleic acid sequencesencoding at least one light chain and nucleic acid sequences encoding atleast two different heavy chains, wherein the heavy and light chains arecapable of pairing with each other; (ii) culturing at least one clone ofthe host cell under conditions conducive to expression of the nucleicacid sequences; (iii) screening at least one clone of the host cell forproduction of a mixture of antibodies having the desired effect by afunctional assay; and (iv) identifying at least one clone that producesa mixture of antibodies having the desired effect. This method, as usedherein, can be performed using high-throughput procedures if desired.The clones identified by the method can be used to produce antibodymixtures according to the invention.

In certain embodiments, the invention further provides transgenicnon-human animals and transgenic plants or transgenic plant cellscapable of expressing mixtures of antibodies and mixtures of antibodiesproduced by these.

In certain embodiments, the invention further provides pharmaceuticalcompositions comprising a mixture of recombinantly produced antibodiesand a suitable carrier.

In certain embodiments, the invention further provides mixtures ofantibodies for use in the treatment or diagnosis and for the preparationof a medicament for use in the treatment or diagnosis of a disease ordisorder in a human or animal subject.

In certain embodiments, the invention further provides a method forproducing a mixture of antibodies comprising different isotypes from asingle host cell clone.

In certain embodiments, the invention further provides a method foridentifying a mixture of antibodies having a desired effect in afunctional assay.

In certain embodiments, the invention further provides a method forproducing a mixture of antibodies that are capable of binding to atarget, the method comprising: i) bringing a phage library comprisingantibodies into contact with material comprising a target, ii) at leastone step of selecting phages binding to the target, iii) identifying atleast two phages that comprise antibodies binding to the target, whereinat least two antibodies comprise a common light chain, iv) introducing anucleic acid sequence encoding the light chain and a nucleic acidsequence or sequences encoding the heavy chains of at least twoantibodies into a host cell, v) culturing a clone of the host cell underconditions conducive to expression of the nucleic acid sequences.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of an antibody. The heavy and lightchains are paired via interchain disulfide bonds (dotted lines). Theheavy chain can be either of the α, γ, μ, δ or ε isotype. The lightchain is either λ or κ. An antibody of IgG1 isotype is shown.

FIG. 2 is a schematic representation of a bispecific monoclonalantibody. A bispecific antibody contains two different functional F(Ab)domains, indicated by the different patterns of the V_(H)-V_(L) regions.

FIGS. 3A and 3B show a sequence alignment of V_(L) (FIG. 3A) and V_(H)(FIG. 3B) of K53, UBS-54 and 02-237. The DNA sequence of common V_(L) ofUBS54 and K53 is SEQ ID NO:1, while the amino acid sequence is given asSEQ ID NO:2. DNA sequences of V_(L) of 02-237, V_(H) of UBS54, K53 and02-237 are SEQ ID NOS:3, 5, 7 and 9, respectively, while the amino acidsequences are given in SEQ ID NOS:4, 6, 8 and 10, respectively.

FIG. 4 is an overview of plasmids pUBS3000Neo and pCD46_(—)3000 (Neo).

FIG. 5, Panel A, shows the isoelectric focusing (IEF) of transientlyexpressed pUBS3000Neo, pCD46_(—)3000(Neo) and a combination of both. InPanel B, the upper part shows a schematic representation of the expectedmolecules when a single light chain and a single heavy chain areexpressed in a cell, leading to monoclonal antibodies UBS-54 or K53. Thelower part under the arrow shows a schematic representation of thecombinations produced when both heavy chains and the common light chainare co-expressed in a host cell, with theoretical amounts when bothheavy chains are expressed at equal levels and pair to each other withequal efficiency. The common light chain is indicated with thevertically striped bars.

FIG. 6 is a schematic representation of a possible embodiment of themethod according to the invention (see, e.g., Example 9). At (1),introduction of nucleic acid sequences encoding one light chain andthree different heavy chains capable of pairing to the common lightchain to give functional antibodies into host cells is shown; at (2),selection of stable clones; (3) shows clones can be screened for, forinstance, expression levels, binding; at (4), clones are expanded; andat (5), production of functional mixtures of antibodies is shown. Someor all of steps 2-5 could be performed simultaneously or in a differentorder.

FIGS. 7A and 7B show the sequence of V_(H) and V_(L) of phages directedagainst CD22 (clone B28), CD72 (clone II-2) (FIG. 7A), and HLA-DR (classII; clone I-2) (FIG. 7B). DNA sequences of V_(L) of clones B28, II-2 and1-2 are SEQ ID NOS:11, 13 and 15, respectively, while the amino acidsequences are SEQ ID NOS:12, 14 and 16, respectively. DNA sequence ofthe common light chain of these clones is SEQ ID NO:17, while the aminoacid sequence is SEQ ID NO:18.

FIG. 8 is a map of pUBS54-IgA (pCRU-L01 encoding human IgA1 againstEPCAM).

FIG. 9 shows dimeric bispecific IgA with a single light chain (indicatedby horizontally striped bar). The method of the invention will produce amixture of forms wherein different heavy chains can be paired. Only themost simple form is depicted in this schematic representation. A J-chainis shown to join the two monomers.

FIG. 10 is a pentameric multispecific IgM with a single light chain(indicated by horizontally striped bars). The method of the inventionwill produce a mixture of many different forms, wherein different heavychains can be paired. Only the most simple form is depicted in thisschematic representation when five different heavy chains are expressedwith a single light chain and all five different heavy chains areincorporated in the pentamer and paired to the same heavy chain.Pentamers with less specificities can also be formed by incorporation ofless than five different heavy chains. Hexamers can also be obtained,especially when the J-chain is not expressed.

FIG. 11 depicts expression of a mixture of human IgG isotypes consistingof a common light chain but with different binding specificities in asingle cell to avoid the formation of bispecific antibodies. Thedifferent binding specificities are indicated by the different colors ofthe V_(H) sequences. The common light chain is indicated with thevertically striped bars. The IgG1 isotype is indicated with the grey Fcand the IgG3 isotype is indicated with the black Fc part.

FIGS. 12A-12E depict DNA and protein sequences of variable domains ofheavy chains of K53 (FIG. 12A), UBS54 (FIG. 12C) and 02-237 (FIG. 12B)IgG (SEQ ID NOS:7, 9 and 5, respectively) and light chains (SEQ ID NOS:1and 3, respectively, for K53/UBS54 (FIG. 12D) and 02-237 IgG (FIG.12E)).

FIG. 13 shows alignment of the variable sequences of the heavy chains ofK53, 02-237 and UBS54 (SEQ ID NOS:7, 9, and 5, respectively). CDR1, CDR2and CDR3 regions are indicated in bold.

FIG. 14 is a BIACORE™ (surface plasmon resonance) analysis of K53 and02-237. Affinity-purified human CD46 from LS174T cells was coupled (640RU) to CM5 chips (BIACORE BR-1000-14™). Binding of 1000 (A), 500 (B),250 (C), 125 (D), 63 (E), 31 (F), 16 (G), 8 (H) or 0 (I) nM 02-237 orK53 purified from stable PER.C6® (human retina cells that expressadenovirus E1A and E1B proteins)-derived cell lines to the CD46 wasmonitored using a BIACORE 3000™ system at 37° C. Using this experimentalset-up, a K_(d) of 9.1×10⁷ and 2.2×10⁸ was found for K53 and 02-237,respectively.

FIG. 15 shows binding of K53 and 02-237 to LS174T cells. Serialdilutions of purified 02-237 (▪), K53 (*) and the negative controlGBSIII (⋄) conjugated to biotin were incubated with LS147T cellspreincubated with normal human serum to block Fcγ receptor interaction.Binding (MFI, ordinate) was determined by FACS after incubation withstreptavidin-conjugated phycoerythrin.

FIG. 16A is an SDS-PAGE analysis of purified IgG fractions. Three μgpurified IgG was analyzed on a non-reduced 4-20% NUPAGE® gel (Novex)according to recommendations of the manufacturer. Proteins werevisualized by staining with colloidal blue (Novex Cat. No LC6025)according to recommendations of the manufacturer. Clone identity isindicated on top of the SDS-PAGE. Each gel contains a control, which iseither purified 02-237 or K53. FIGS. 16B and 16C are continuations ofthe gel in FIG. 16A.

FIG. 16D is an SDS-PAGE analysis of purified IgG fractions. Three μgpurified IgG was analyzed on a reduced 4-20% NUPAGE® gel according torecommendations of the manufacturer. Proteins were visualized bystaining with colloidal blue (Novex cat. No LC6025) according torecommendations of the manufacturer. Clone identity is indicated on topof the SDS-PAGE. Each gel contains a control, which is either purified02-237 or K53. NR, Non-reduced; R, reduced. FIGS. 16E and 16F arecontinuations of the gel in FIG. 16D.

FIG. 17A shows an IEF analysis of purified IgG fractions. Ten μgpurified IgG was analyzed on an Isogel 3-10 gel (BMA) according torecommendations of the manufacturer. Proteins were visualized bystaining with colloidal blue according to recommendations of themanufacturer. Clone identity is indicated on top of the IEF. Each gelcontains a control, consisting of a 1:1:1 mixture of 02-237, K53 andUBS54. FIGS. 17B through 17D are continuations of the gel in FIG. 17A.

FIG. 18 is an IEF analysis of polyclonal mixtures 241, 280, 282, 361 and402 in comparison to single K53, 02-237 and UBS54. Ten μg purified IgGwas analyzed on an Isogel 3-10 gel (BMA) according to recommendations ofthe manufacturer. Proteins were visualized by staining with colloidalblue according to recommendations of the manufacturer. IgG identity isindicated on top of the IEF.

FIG. 19 contains mass chromatograms of CDR3 peptides of K53, 02-237,UBS54 and the two unique light chain peptides L1-K53/UBS54 and L1-237 inIgG fraction Poly1-280. On the right-hand side of each masschromatogram, the isotopic pattern of the peptide is shown. The doublycharged ion at m/z 1058.98 (Mw 2115.96 Da) results from peptide H11-K53.The doubly charged ion at m/z 1029.96 (Mw 2057.92 Da) results frompeptide H11-02-237. The triply charged ion at m/z 770.03 (Mw 2307.09 Da)results from peptide H9-UBS54. The doubly charged ion at m/z 1291.08 (Mw2580.16 Da) results from peptide L1-K53/UBS54. The doubly charged ion atm/z 1278.11 (Mw 2554.22 Da) results from peptide L1-02-237.

Purified IgG was dissolved in a 0.1% RAPIGEST™ (Waters) in 50 mMNH₄HCO₃. The disulfides were reduced using 1 M DTT(1,4-dithio-DL-threitol), followed by incubation at 65° C. for 30minutes. Then, for alkylation of all sulfhydryl groups, 1 Miodoacetamide was added, followed by incubation at room temperature for45 minutes in the dark. Alkylation was stopped by addition of 1 M DTT.The buffer was exchanged to 25 mM NH₄HCO₃, pH 7.5. Finally, theantibodies were digested overnight at 37° C. by addition of a freshlyprepared trypsin solution in 25 mM NH₄HCO₃. The peptide mixture wasanalyzed by LC-MS. The LC-system consisted of a Vydac reversed-phase C18column that was eluted by applying a gradient of solvent A (5/95/1acetonitrile, water, glacial acetic acid v/v/v) and solvent B (90/10/1acetonitrile, water, glacial acetic acid v/v/v). The LC was on-linecoupled to a Q-TOF2 mass spectrometer (Micromass), equipped with anelectrospray source operated at 3 kV. Mass spectra were recorded in apositive ion mode from m/z 50 to 1500 at a cone voltage of 35 V. Theinstrumental resolution of >10,000 enabled unambiguous determination ofthe charge and, therefore, the mass of most ions up to at least +7. Inthis way, all peptides were identified according to their molecularweight. The amino acid sequence of the peptide was confirmed byMS/MS-experiments. MS/MS spectra were recorded in a positive ion modefrom m/z 50-2000 with collision energy between 20 and 35 eVolts.

FIG. 20 is a BIACORE™ (surface plasmon resonance) analysis of polyclonal280. Affinity-purified human CD46 from LS174T cells was coupled (640 RU)to CM5 chips (BIACORE BR-1000-14™). Binding of 1000 (A), 500 (B), 250(C), 125 (D), 63 (E), 31 (F), 16 (G), 8 (H) or 0 (I) nM Poly1-280 toCD46 was monitored using a BIACORE 3000™ system at 37° C.

FIG. 21 is an IEF analysis of sub-clones from clones poly 1-241, poly1-280 and poly 1-402 producing a mixture of antibodies.

Panel A contains clones poly 1-241 and poly 1-280. Lane 1 contains a pImarker (Amersham, Cat. No. 17-0471-01). Lane 2 contains isolated IgGfrom the parent clone poly 1-241 (as in FIG. 18). Lanes 3, 4 and 5,respectively, contain isolated IgG from three independent sub-clonesderived from poly 1-241 by limiting dilution. Lane 6 contains isolatedIgG from the parent clone poly 1-280 (as in FIG. 18). Lanes 7, 8 and 9,respectively, contain isolated IgG from three independent sub-clonesderived from poly 1-280 by limiting dilution.

Panel B contains clone poly 1-402. Lanes 1 and 7 contain a pI marker.Lane 2 contains isolated IgG from the parent clone poly 1-402 (as inFIG. 18). Lanes 3, 4 and 5, respectively, contain isolated IgG fromthree independent sub-clones derived from poly 1-402 by limitingdilution. Lane 6 contains a control (a 1:1:1 mixture of 02-237, K53 andUBS54).

FIG. 22 is a fluorescence activated cell sorting (FACS) analysis ofmixtures of antibodies produced from sub-clones of poly 1-241 (A), poly1-280 (B) and poly 1-402 (C). Binding of the mixtures of antibodies tocells transfected with cDNA of CD46, EpCAM, or a negative control(CD38), was determined with FACS analysis. Mean fluorescent intensity(MFI) is shown for the various parent clones and three independentsub-clones of each. Control antibodies GBS-III (negative control),anti-CD72 (02-004; negative control) and the single antibodies UBS54,02-237 and K53 are also included.

DETAILED DESCRIPTION OF THE INVENTION

In certain embodiments, also provided is a method for producing amixture of antibodies in a recombinant host, the method comprisingexpressing, in a recombinant host cell, a nucleic acid sequence ornucleic acid sequences encoding at least one light chain and at leastthree different heavy chains that are capable of pairing with at leastone light chain. In certain embodiments, the light and heavy chains formfunctional antigen-binding domains when paired. A functionalantigen-binding domain is capable of specifically binding to an antigen.

In certain embodiments, the method for producing a mixture of antibodiesaccording to the invention further comprises the step of recovering theantibodies from the cell or the host cell culture to obtain a mixture ofantibodies suitable for further use.

In certain embodiments, a method is provided for production of a mixtureof antibodies, the method comprising expressing in a recombinant hostcell a nucleic acid sequence encoding a common light chain and nucleicacid sequence or sequences encoding at least three different heavychains that are capable of pairing with the common light chain, suchthat the antibodies that are produced comprise common light chains. Inone aspect, the common light chain is identical in each lightchain/heavy chain pair.

The term “antibody,” as used herein, means a polypeptide containing oneor more domains that bind an epitope on an antigen, where such domainsare derived from, or have sequence identity with, the variable region ofan antibody. The structure of an antibody is schematically representedin FIG. 1. Examples of antibodies according to the invention includefull length antibodies, antibody fragments, bispecific antibodies,immunoconjugates, and the like. An antibody, as used herein, may beisotype IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, IgM, and the like,or a derivative of these. Antibody fragments include Fv, Fab, Fab′,F(ab′)₂ fragments, and the like. Antibodies according to the inventioncan be of any origin, including murine, of more than one origin, e.g.,chimeric, humanized, or fully human antibodies. Immunoconjugatescomprise antigen-binding domains and a non-antibody part such as atoxin, a radiolabel, an enzyme, and the like.

An “antigen-binding domain” preferably comprises variable regions of aheavy and a light chain and is responsible for specific binding to anantigen of interest. Recombinant antibodies are prepared by expressingboth a heavy and a light chain in a host cell. Similarly, by expressingtwo chains with their respective light chains (or a common light chain),wherein each heavy chain/light chain has its own specificity, so-called“bispecific” antibodies can be prepared. “Bispecific antibodies”comprise two non-identical heavy-light chain combinations (FIG. 2), andboth antigen-binding regions of a bispecific antibody may recognizedifferent antigens or different epitopes on an antigen. “Epitope” meansa moiety of an antigen to which an antibody binds. A single antigen mayhave multiple epitopes.

A “common light chain,” refers to light chains which may be identical orhave amino acid sequence differences. Common light chains may comprisemutations which do not alter the specificity of the antibody whencombined with the same heavy chain without departing from the scope ofthe invention. It is, for instance, possible within the scope of thedefinition of common light chains as used herein, to prepare or findlight chains that are not identical but still functionally equivalent,e.g., by introducing and testing conservative amino acid changes,changes of amino acids in regions that do not or only partly contributeto binding specificity when paired with the heavy chain, and the like.In an exemplary embodiment, the invention provides the use of a commonlight chain, one identical light chain, to combine with different heavychains to form antibodies with functional antigen-binding domains. Theuse of one common light chain avoids the formation of heterodimers inwhich pairing of light and heavy chains results in antigen-bindingdomains that are not functional or, in other words, which are notcapable of binding to the target antigen or antigens. The use of acommon light chain and two heavy chains has been proposed (Merchant etal., 1998; WO 98/50431, the entirety of which are incorporated herein byreference) for a different purpose, viz., to increase the formation offunctional bispecific antibodies at the expense of antibody mixturecomplexity. These publications teach a method for preferentiallyproducing one defined and desired bispecific antibody, therebyminimizing the complexity of the produced mixture. Hence, Merchantspecifically teaches to prevent the production of monospecificantibodies because these are undesired byproducts in the process forbispecific antibody production described in those publications. Clearly,there is no teaching in the prior art to prepare a complex mixture ofantibodies from a recombinant host cell avoiding the formation ofnon-functional binding domains or the benefits thereof, let alone how.In the method according to the invention, at least three different heavychains that are capable of pairing with the common light chain areexpressed. In other embodiments, the host cell, as used herein, isprovided with nucleic acid sequences encoding for 4, 5, 6, 7, 8, 9, 10,or more, heavy chains capable of pairing with the common light chain, toincrease the complexity of the produced mixture of antibodies.

“Different heavy chains,” according to the invention, may differ in thevariable region and have the same constant region. In other embodiments,where it is clear from the context, they may have the same variableregion and differ in the constant region, e.g., be of a differentisotype. The use of a mixture of antibodies having different constantregions, such as the Fc-portion, may be advantageous if different armsof the immune system are to be mobilized in the treatment of the humanor animal body. In yet other embodiments, also to be clear from thecontext, both the variable and the constant regions may differ.

A “mixture of antibodies,” according to the invention, comprises atleast two non-identical antibodies, but may comprise 3, 4, 5, 6, 7, 8,9, 10, or more, different antibodies and may resemble a polyclonal or atleast an oligoclonal antibody mixture with regard to complexity andnumber of functional antigen-binding molecules. The mixtures producedaccording to the invention usually will comprise bispecific antibodies.If desired, formation of monospecific antibodies in the mixture can befavored over the formation of bispecific antibodies.

When n heavy chains and one common light chain are expressed, as usedherein, in a host cell at equal levels, the theoretical percentage ofbispecific antibodies produced by the method according to the inventionis (1−1/n)×100%. The total number of different antibodies in the mixtureproduced by the method according to the invention is theoreticallyn+{(n²−n)/2}, of which (n²−n/2) are bispecific antibodies. Distortion ofthe ratio of expression levels of the different heavy chains may lead tovalues deviating from the theoretical values. The amount of bispecificantibodies can also be decreased, compared to these theoretical values,if all heavy chains do not pair with equal efficiency. It is, forinstance, possible to engineer the heavy chains, for example, byintroducing specific and complementary interaction surfaces betweenselected heavy chains, to promote homodimer pairing over heterodimerpairing, contrary to what has been proposed by Merchant, supra. Heavychains may also be selected so as to minimize heterodimer formation inthe mixture. A special form of this embodiment involves heavy chains oftwo or more different isotypes (e.g., IgG1, IgG3, IgA). When heavychains of different isotype are expressed in the same host cell inaccordance with the invention and one light chain that can pair to theseheavy chains, the amount of bispecific antibodies will be reduced,possibly to very low or even undetectable levels. Thus, when bispecificantibodies are less desirable, it is possible to produce a mixture ofantibodies according to the invention, wherein a nucleic acid sequenceencoding a common light chain and nucleic acid sequences encoding atleast two different heavy chains with a different variable regioncapable of pairing to the common light chain are expressed in arecombinant host, and wherein the heavy chains further differ in theirconstant regions sufficiently to reduce or prevent pairing between thedifferent heavy chains. The mixtures of antibodies may be produced froma clone that was derived from a single host cell, i.e., from apopulation of cells containing the same recombinant nucleic acidsequences.

It will be understood that the different heavy chains can be encoded onseparate nucleic acid molecules, but may also be present on one nucleicacid molecule comprising different regions encoding at least three heavychains. The nucleic acid molecules usually encode precursors of thelight and/or heavy chains, which, when expressed, are secreted from thehost cells, thereby becoming processed to yield the mature form. Theseand other aspects of expressing antibodies in a host cell are well knownto those having ordinary skill in the art.

A “recombinant host cell,” as used herein, is a cell comprising one ormore so-called transgenes, i.e., recombinant nucleic acid sequences notnaturally present in the cell. These transgenes are expressed in thehost cell to produce recombinant antibodies encoded by these nucleicacid sequences when these cells are cultured under conditions conduciveto expression of nucleic acid sequences. The host cell, as used herein,can be present in the form of a culture from a clone that is derivedfrom a single host cell wherein the transgenes have been introduced. Toobtain expression of nucleic acid sequences encoding antibodies, it iswell known to those skilled in the art that sequences capable of drivingsuch expression can be functionally linked to the nucleic acid sequencesencoding the antibodies.

“Functionally linked” is meant to describe that the nucleic acidsequences encoding the antibody fragments or precursors thereof islinked to the sequences capable of driving expression such that thesesequences can drive expression of the antibodies or precursors thereof.

Useful expression vectors are available in the art, for example, thepcDNA vector series of Invitrogen. Where the sequence encoding thepolypeptide of interest is properly inserted with reference to sequencesgoverning the transcription and translation of the encoded polypeptide,the resulting expression cassette is useful to produce the polypeptideof interest, referred to as expression. Sequences driving expression mayinclude promoters, enhancers and the like, and combinations thereof.These should be capable of functioning in the host cell, thereby drivingexpression of the nucleic acid sequences that are functionally linked tothem. Promoters can be constitutive or regulated and can be obtainedfrom various sources, including viruses, prokaryotic or eukaryoticsources, or artificially designed. Expression of nucleic acids ofinterest may be from the natural promoter or derivative thereof or froman entirely heterologous promoter. Some well-known and much-usedpromoters for expression in eukaryotic cells comprise promoters derivedfrom viruses, such as adenovirus, for instance, the E1A promoter,promoters derived from cytomegalovirus (CMV), such as the CMV immediateearly (IE) promoter, promoters derived from Simian Virus 40 (SV40), andthe like. Suitable promoters can also be derived from eukaryotic cells,such as methallothionein (MT) promoters, elongation factor 1α (EF-1α)promoter, an actin promoter, an immunoglobulin promoter, heat shockpromoters, and the like. Any promoter or enhancer/promoter capable ofdriving expression of the sequence of interest in the host cell issuitable in the invention. In one embodiment, the sequence capable ofdriving expression comprises a region from a CMV promoter, preferablythe region comprising nucleotides −735 to +95 of the CMV immediate earlygene enhancer/promoter. The skilled artisan will be aware that theexpression sequences used in the invention may suitably be combined withelements that can stabilize or enhance expression, such as insulators,matrix attachment regions, STAR elements (WO 03/004704, the entirety ofwhich is incorporated herein by reference), and the like. This mayenhance the stability and/or levels of expression.

Protein production in recombinant host cells has been extensivelydescribed, e.g., in Current Protocols in Protein Science, 1995, ColiganJ. E., Dunn B. M., Ploegh H. L., Speicher D. W., Wingfield P. T., ISBN0-471-11184-8; Bendig, 1988, the entirety of which is incorporatedherein by reference. Culturing a cell is done to enable it tometabolize, grow, divide, and/or produce recombinant proteins ofinterest. This can be accomplished by methods well known to personsskilled in the art and includes, but is not limited to, providingnutrients for the cell. The methods comprise growth adhering tosurfaces, growth in suspension, or combinations thereof. Severalculturing conditions can be optimized by methods well known in the artto optimize protein production yields. Culturing can be done, forinstance, in dishes, roller bottles or in bioreactors, using batch,fed-batch, continuous systems, hollow fiber, and the like. In order toachieve large-scale (continuous) production of recombinant proteinsthrough cell culture, it is preferred in the art to have cells capableof growing in suspension and it is preferred to have cells capable ofbeing cultured in the absence of animal- or human-derived serum oranimal- or human-derived serum components. Thus, purification is easierand safety is enhanced due to the absence of additional animal or humanproteins derived from the culture medium, while the system is also veryreliable as synthetic media are the best in reproducibility.

“Host cells,” according to the invention, may be any host cell capableof expressing recombinant DNA molecules, including bacteria such asEscherichia (e.g., E. coli), Enterobocter, Salmonella, Bacillus,Pseudomonas, Streptomyces, yeasts such as S. cerevisiae, K lactis, P.pastoris, Candida, or yarrowia, filamentous fungi such as Neurospora,Aspergillus oryzae, Aspergillus nidulans and Aspergillus niger, insectcells such as Spodoptera frugiperda SF-9 or SF-21 cells, mammalian cellssuch as Chinese hamster ovary (CHO) cells, BHK cells, mouse cellsincluding SP2/0 cells and NS-0 myeloma cells, primate cells such as COSand Vero cells, MDCK cells, BRL 3A cells, hybridomas, tumor cells,immortalized primary cells, human cells such as W138, HepG2, HeLa,HEK293, HT1080 or embryonic retina cells such as PER.C6® (human retinacells that express adenovirus E1A and E1B proteins), and the like.Often, the expression system of choice will involve a mammalian cellexpression vector and host so that the antibodies are appropriatelyglycosylated. A human cell line, preferably PER.C6® (human retina cellsthat express adenovirus E1A and E1B proteins), can advantageously beused to obtain antibodies with a completely human glycosylation pattern.The conditions for growing or multiplying cells (see, e.g., TissueCulture, Academic Press, Kruse and Paterson, editors (1973), theentirety of which is incorporated herein by reference) and theconditions for expression of the recombinant product may differ somewhatand optimization of the process is usually performed to increase theproduct yields and/or growth of the cells with respect to each other,according to methods generally known to one of ordinary skill in theart.

In general, principles, protocols, and practical techniques formaximizing the productivity of mammalian cell cultures can be found inMammalian Cell Biotechnology: a Practical Approach (M. Butler, ed., IRLPress, 1991), the entirety of which is incorporated herein by reference.Expression of antibodies in recombinant host cells has been extensivelydescribed in the art (see, e.g., EP0120694; EP0314161; EP0481790;EP0523949; U.S. Pat. No. 4,816,567; WO 00/63403, the entirety of whichare incorporated herein by reference). The nucleic acid moleculesencoding the light and heavy chains may be present as extrachromosomalcopies and/or stably integrated into the chromosome of the host cell.With regard to stability of production, the latter is preferred.

The antibodies are expressed in the cells according to the invention andmay be recovered from the cells or, preferably, from the cell culturemedium, by methods generally known to persons skilled in the art. Suchmethods may include precipitation, centrifugation, filtration,size-exclusion chromatography, affinity chromatography, cation- and/oranion-exchange chromatography, hydrophobic interaction chromatography,and the like. For a mixture of antibodies comprising IgG molecules,protein A- or protein G-affinity chromatography can be suitably used(see, e.g., U.S. Pat. Nos. 4,801,687 and 5,151,504, the entirety ofwhich are incorporated herein by reference).

In one embodiment, at least two antibodies from the mixture producedaccording to the invention comprise a heavy-light chain dimer havingdifferent specificities and/or affinities. The specificity determineswhich antigen or epitope thereof is bound by the antibody. The affinityis a measure for the strength of binding to a particular antigen orepitope. Specific binding is defined as binding with an affinity (K_(a))of at least 5×10⁴ liter/mole, more preferably, 5×10⁵, even morepreferably, 5×10⁶, and still more preferably, 5×10⁷, or more. Typically,monoclonal antibodies may have affinities which go up to 10¹⁰ liter permole or even higher. The mixture of antibodies produced according to theinvention may contain at least two antibodies that bind to differentepitopes on the same antigen molecule and/or may contain at least twoantibodies that bind to different antigen molecules present in oneantigen-comprising mixture. Such an antigen-comprising mixture may be amixture of partially or wholly purified antigens, such as toxins,membrane components and proteins, viral envelope proteins, or it may bea healthy cell, a diseased cell, a mixture of cells, a tissue or mixtureof tissues, a tumor, an organ, a complete human or animal subject, afungus or yeast, a bacteria or bacterial culture, a virus or virusstock, or combinations of these, and the like. Unlike monoclonalantibodies that are able to bind to a single antigen or epitope only,the mixture of antibodies according to the invention may, therefore,have many of the advantages of a polyclonal or oligoclonal antibodymixture.

In a preferred embodiment, the host cell according to the method of theinvention is capable of high-level expression of human immunoglobulin,i.e., at least 1 picograms per cell per day, preferably, at least 10picograms per cell per day and, even more preferably, at least 20picograms per cell per day or more without the need for amplification ofthe nucleic acid molecules encoding the heavy and light chains in thehost cell.

Preferably, host cells according to the invention contain in theirgenome between one and ten copies of each recombinant nucleic acid to beexpressed. In the art, amplification of the copy number of the nucleicacid sequences encoding a protein of interest in, e.g., CHO cells can beused to increase expression levels of the recombinant protein by thecells (see, e.g., Bendig, 1988; Cockett et al., 1990; U.S. Pat. No.4,399,216, the entirety of which are incorporated herein by reference).This is currently a widely used method. However, a significanttime-consuming effort is required before a clone with a desired highcopy number and high expression levels has been established and,moreover, clones harboring very high copy numbers (up to hundreds) ofthe expression cassette often are unstable (e.g., Kim et al., 1998, theentirety of which is incorporated herein by reference). It is,therefore, a preferred embodiment of the invention to use host cellsthat do not require such amplification strategies for high-levelexpression of the antibodies of interest. This allows fast generation ofstable clones of host cells that express the mixture of antibodiesaccording to the invention in a consistent manner. We provide evidencethat host cells according to the invention can be obtained, sub-clonedand further propagated for at least around 30 cell divisions (populationdoublings) while expressing the mixture of antibodies according to theinvention in a stable manner, in the absence of selection pressure.Therefore, in certain aspects, the methods of the invention includeculturing the cells for at least 20, preferably 25, more preferably 30,population doublings and, in other aspects, the host cells according tothe invention have undergone at least 20, preferably 25, more preferably30, population doublings and are still capable of expressing a mixtureof antibodies according to the invention. Also provided is a culture ofcells producing a mixture of immunoglobulins from a single cell, themixture comprising at least three different heavy chains. Also providedis a culture of cells producing at least three different monospecificimmunoglobulins from a single cell. In certain exemplary aspects, theculture produces the mixture or at least three different monospecificimmunoglobulins in a single cell for more than 20, preferably more than25, more preferably, more than 30 population doublings.

Preferably, host cells according to the method of the invention arederived from human retina cells that have been immortalized ortransformed with adenoviral E1 sequences. A particularly preferred hostcell according to methods of the invention is PER.C6® (human retinacells that express adenovirus E1A and E1B proteins) as deposited underECACC no. 96022940, or a derivative thereof. PER.C6-derived clones canbe generated fast, usually contain a limited number of copies (about1-10) of the transgene, and are capable of high-level expression ofrecombinant antibodies (Jones et al., 2003, the entirety of which isincorporated herein by reference). Therefore, such clones are expectedto maintain a stable copy number over many generations, which is anadvantage in the production of biopharmaceuticals. PER.C6® (human retinacells that express adenovirus E1A and E1B proteins) cells have beenextensively characterized and documented, demonstrating good process ofscaling up, suspension growth and growth factor independence.Furthermore, PER.C6® (human retina cells that express adenovirus E1A andE1B proteins) can be incorporated into a suspension in a highlyreproducible manner, making it particularly suitable for large-scaleproduction. In this regard, the PER.C6® cell line (human retina cellsthat express adenovirus E1A and E1B proteins) has been characterized forbioreactor growth, where it can grow to very high densities. The use ofPER.C6® (human retina cells that express adenovirus E1A and E1Bproteins) for recombinant production of antibodies has been described indetail in publication WO 00/63403 and in (Jones et al., 2003, theentirety of which is incorporated herein by reference).

Also provided is a mixture of antibodies obtainable by a methoddescribed herein. Such mixtures of antibodies are expected to be moreeffective than the sole components it comprises, in analogy topolyclonal antibodies usually being more effective than monoclonalantibodies to the same target. Such mixtures can be prepared against avariety of target antigens or epitopes.

It certain embodiments, the invention provides a recombinant host cellcomprising a nucleic acid sequence encoding a light chain and a nucleicacid sequence or nucleic acid sequences encoding at least threedifferent heavy chains of an antibody, wherein the light chain and heavychains are capable of pairing, preferably to form a functional bindingdomain. The paired heavy and light chains form functionalantigen-binding regions against the target antigen or target antigens.The host cells are useful in the described methods. They can be used toproduce mixtures of antibodies.

In certain embodiments, the invention provides a composition comprisinga mixture of recombinantly produced antibodies, wherein at least threedifferent heavy chain sequences are represented in the mixture ofrecombinant antibodies. Monoclonal antibodies are routinely produced byrecombinant methods. Also disclosed are mixtures of antibodies usefulfor diagnosis or treatment in various fields. In certain embodiments,the compositions of the invention comprise mixtures of at least threedifferent heavy chains paired to light chains in the form of antibodies.Preferably, the light chains of the antibodies in the mixtures have acommon light chain. The mixtures may comprise bispecific antibodies. Themixtures may be produced from a clone that was derived from a singlehost cell, e.g., from a population of cells containing the samerecombinant nucleic acid sequences. The mixtures can be obtained bymethods according to the invention or be produced by host cellsaccording to the invention. In other embodiments, the number of heavychains represented in the mixture is 4, 5, 6, 7, 8, 9, 10, or more. Theoptimal mixture for a certain purpose may be determined empirically bymethods known to one of ordinary skill in the art or by methods providedby the invention. Such compositions according to the invention may haveseveral of the advantages of a polyclonal antibody mixture, without thedisadvantages usually inherently associated with polyclonal antibodymixtures, because of the manner in which they are produced. It isfurthermore expected that the mixture of antibodies is more efficaciousthan separate monoclonal antibodies. Therefore, the dosage and, hence,the production capacity required may be less for the mixtures ofantibodies according to the invention than for monoclonal antibodies.

It has, for instance, been described that although no single monoclonalantibody to botulinum neurotoxin (BoNTIA) significantly neutralizedtoxin, a combination of three such monoclonal antibodies (oligoclonalantibody) neutralized 450,000 50% lethal doses of BoNTIA, a potency 90times greater than human hyperimmune globulin (Nowakowski et al., 2002,the entirety of which is incorporated herein by reference). This resultdemonstrates that oligoclonal mixtures of antibodies comprising only twoto three different specificities may have very high potency.

Furthermore, the chances of a mixture of the invention losing itsactivity due to target or epitope loss is reduced, when compared to asingle monoclonal antibody. In particular embodiments, 2, 3, 4, 5, 6, 7,8, 9, 10, or more of the antibodies present in the mixture according tothe invention have different specificities. Different specificities maybe directed to different epitopes on the same antigen and/or may bedirected to different antigens present in one antigen-comprisingmixture. A composition according to the invention may also furthercomprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antibodies having differentaffinities for the same epitope. Antibodies with differing affinitiesfor the same epitope may, for instance, be generated by methods ofaffinity maturation known to one of ordinary skill in the art.

In a particularly preferred embodiment, the composition according to theinvention has an effect that is greater than the effect of eachindividual monospecific antibody present in the composition. The effectcan be measured in a functional assay. A “functional assay,” as usedherein, is an assay that can be used to determine one or more desiredparameters of the antibody or the mixture of antibodies subject to theassay conditions. Suitable functional assays may be binding assays,apoptosis assays, antibody-dependent cellular cytotoxicity (ADCC)assays, complement-dependent cytotoxicity (CDC) assays, inhibition ofcell growth or proliferation (cytostatic effect) assays, cell-killing(cytotoxic effect) assays, cell-signaling assays, assays for measuringinhibition of binding of pathogen to target cell, assays to measure thesecretion of vascular endothelial growth factor (VEGF) or other secretedmolecules, assays for bacteriostasis, bactericidal activity,neutralization of viruses, assays to measure the attraction ofcomponents of the immune system to the site where antibodies are bound,including in situ hybridization methods, labeling methods, and the like.Clearly, also in vivo assays, such as animal models, including mousetumor models, models of auto-immune disease, virus-infected orbacteria-infected rodent or primate models, and the like, can be usedfor this purpose. The efficacy of a mixture of antibodies according tothe invention can be compared to individual antibodies in such models bymethods generally known to one of ordinary skill in the art.

In certain embodiments, the invention provides a method for identifyingat least one host cell clone that produces a mixture of antibodies,wherein the mixture of antibodies has a desired effect according to afunctional assay, the method comprising (i) providing a host cellcomprising a nucleic acid sequence encoding at least one light chain andnucleic acid sequence or sequences encoding at least two different heavychains, wherein the heavy and light chains are capable of pairing witheach other; (ii) culturing at least one clone of the host cell underconditions conducive to expression of nucleic acid sequences; (iii)screening at least one clone of the host cell for production of amixture of antibodies having the desired effect by a functional assay;and (iv) identifying at least one clone that produces a mixture ofantibodies having the desired effect. Preferably, the host cellcomprises a nucleic acid sequence encoding a common light chain that iscapable of pairing with at least two different heavy chains, such thatproduced antibodies comprise common light chains, as described above. Inspecific embodiments, culturing in step (ii) and screening in step (iii)of the method is performed with at least two clones. The method mayoptionally include an assay for measuring the expression levels of theantibodies that are produced, which assay may be during or after step(ii) according to the method, or later in the procedure. Such assays arewell known to one of ordinary skill in the art and include proteinconcentration assays, immunoglobulin-specific assays such as ELISA, RIA,DELFIA, and the like. In particular embodiments of the method accordingto the invention, the host cell comprises nucleic acid sequence orsequences encoding at least 3, 4, 5, 6, 7, 8, 9, 10, or more, heavychains capable of pairing with at least one light chain. Functionalassays useful for the method according to the invention may be assaysfor apoptosis, ADCC, CDC, cell killing, inhibition of proliferation,virus neutralization, bacterial opsonization, receptor-mediatedsignaling, cell signaling, bactericidal activity, and the like. Usefulscreening assays for anti-cancer antibodies have, for instance, beendescribed in U.S. Pat. No. 6,180,357, the entirety of which isincorporated herein by reference. Such assays may also be used toidentify a clone according to the method of the invention. It is, forinstance, possible to use enzyme-linked immunosorbent assays (ELISAs)for the testing of antibody binding to their target. Using such assays,it is possible to screen for antibody mixtures that most avidly bind thetarget antigen (or mixture of target antigens against which the mixtureof antibodies is to be tested). Another possibility that can be exploredis to directly screen for cytotoxicity or cytostatic effects. It ispossible that upon such a different screen, other or the same clonesproducing mixtures of antibodies will be chosen than with the ELISAmentioned above. The screening for cell killing or cessation of growthof cancerous cells may be suitably used according to the invention. Celldeath can be measured by various endpoints, including the absence ofmetabolism or the denaturation of enzymes. In one possible embodiment ofthe invention, the assay is conducted by focusing on cytotoxic activitytoward cancerous cells as an endpoint. For this assay, a live/dead assaykit, for example, the LIVE/DEAD® Viability/Cytotoxicity Assay Kit(L-3224) by Molecular Probes (Eugene, Oreg.), can suitably be used.Other methods of assessing cell viability, such as tryspan blueexclusion, ⁵¹Cr release, Calcein-AM, ALAMAR BLUE™, LDH activity, andsimilar methods, can also be used. The assays may also include screeningof the mixture of antibodies for specificity to the desiredantigen-comprising tissue. The antibodies according to the invention mayhave a limited tissue distribution. It is possible to include testingthe mixtures of antibodies against a variety of cells, cell types, ortissues, to screen for mixtures of antibodies that preferably bind tocells, cell types or tissues of interest.

Irrespective of a functional assay as described above, also disclosedherein are ways to determine the identity of the antibodies expressed bya clone, using methods such as iso-electric focusing (IEF),mass-spectrometry (MS), and the like. In certain embodiments, therefore,the invention provides use of MS and/or IEF in selecting a clone thatexpresses a mixture of antibodies according to the invention.

When monoclonal antibodies are produced by recombinant host cells, ascreening step is usually performed to assess expression levels of theindividual clones that were generated. The addition of more heavy chainsto produce mixtures adds a level of complexity to the production ofantibodies. When host cells are transfected with nucleic acid moleculesencoding the light and heavy chains that will form the mixture ofantibodies desired, independent clones may arise containing the samegenetic information but, nevertheless, differing in expression levels,thereby producing different ratios of the encoded antibodies, givingrise to different mixtures of antibodies from the same geneticrepertoire. The method according to the invention is useful foridentifying a clone that produces an optimal mixture for a certainpurpose.

The culturing and/or screening according to steps (ii) and (iii),respectively, may be suitably performed using high-throughputprocedures, optionally in an automated fashion. Clones can, forinstance, be cultured in 96-well plates or other multi-well plates,e.g., in arrayed format, and screened for production of a desiredmixture. Robotics may be suitably employed for this purpose. Methods toimplement high-throughput culturing and assays are generally availableand known to one of ordinary skill in the art. It will also be clearthat for this method according to the invention, it is beneficial to usehost cells capable of high-level expression of proteins, without theneed for amplification of the nucleic acid encoding the proteins in thecell. In one embodiment, the host cell is derived from a human embryonicretinoblast cell that has been immortalized or transformed by adenoviralE1 sequences. In a preferred embodiment, the cell is derived fromPER.C6® (human retina cells that express adenovirus E1A and E1Bproteins). This cell line has already been shown to be amenable tohigh-throughput manipulations, including culturing (WO 99/64582, theentirety of which is incorporated herein by reference).

In specific embodiments of the invention, the mixture of antibodiesaccording to the method of identifying at least one host cell accordingto the invention, comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore, antibodies having different specificities and/or affinities.

A potential advantage of the method will be that it will allow exploringmany possible combinations simultaneously, the combinations inherentlyincluding the presence of bispecific antibodies in the produced mixture.Therefore, more combinations can be tested than by just mixing purifiedknown monoclonal antibodies, both in number of combinations and inratios of presence of different antibodies in these combinations.

The clone that has been identified by the method according to theinvention can be used for producing a desired mixture of antibodies. Incertain embodiments, the invention provides a method of producing amixture of antibodies, the method comprising culturing a host cell cloneidentified by the method of identifying at least one host cell clonethat produces a mixture of antibodies according to the invention,culturing being under conditions conducive to expression of the nucleicacid molecules encoding at least one light chain and at least twodifferent heavy chains. The produced antibodies may be recovered fromthe host cells and/or from the host cell culture, for example, from theculture medium. The mixture of antibodies can be recovered according toa variety of techniques known to one of ordinary skill in the art.

In certain embodiments, the invention provides a mixture of antibodiesobtainable by the method according to the invention described above. Themixtures can be used for a variety of purposes, such as in the treatmentor diagnosis of disease, and may replace, or be used in addition to,monoclonal or polyclonal antibodies.

The methods according to the invention may suitably use nucleic acidmolecules for encoding the antibodies, which nucleic acid molecules havebeen obtained by any suitable method, including in vivo, e.g.,immunization, methods or in vitro, for instance, antibody displaymethods (A. Plückthun et al., In vitro selection and evolution ofproteins, in Adv. Prot. Chem., F. M. Richards et al., Eds, AcademicPress, San Diego, 2001, vol. 55:367-403, the entirety of which isincorporated herein by reference), such as phage display, ribosomedisplay or mRNA display (C. Schaffitzel et al., In vitro selection andevolution of protein-ligand interactions by ribosome display, inProtein-Protein Interactions, A Molecular Cloning Manual, E. Golemis,Ed., Cold Spring Harbor Laboratory Press, New York, 2001, pp. 535-567,the entirety of which is incorporated herein by reference), and yeastdisplay (e.g., WO 99/36569, the entirety of which is incorporated hereinby reference). Methods of identifying antibodies to a certain target,which target may be a known antigen or an unknown antigen present in anantigenic mixture, by phage display are known to one of ordinary skillin the art. In general, a library of phages that express anantigen-binding domain or derivative thereof on their surface, theantigen-binding domain encoded by genetic material present in thephages, is incubated with the antigen or antigen mixture of interest,after which binding of a sub-population of the phages that displayantigen-binding sites binding to the desired antigen is obtained whereasthe non-binding phages are discarded. Such selection steps may berepeated one, two, or more times to obtain a population of phages thatare more or less specific for the antigen of interest. Phage displaymethods to obtain antibodies, parts or derivatives thereof have beenextensively described in C. F. Barbas III et al., Phage Display, Alaboratory manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 2001, the entirety of which is incorporated herein byreference. The library used for such screening may be generated by usingthe genetic information of one or more light chains, combined withgenetic information encoding a plurality of heavy chains. The librarydescribed by De Kruif et al. (1995b), the entirety of which isincorporated herein by reference, comprises seven light chains, theentirety of which is incorporated herein by reference. Therefore, in apanel of phages binding to a target, which can, e.g., be obtained bymethods described in De Kruif et al. (supra), and U.S. Pat. No.6,265,150 (the entirety of which is incorporated herein by reference),not more than seven different light chains will be represented and, ifthe panel is large enough, several phages with the same light chaincoupled to unrelated heavy chains may be found. Such phages can be usedto obtain the nucleic acid molecules useful in the methods according tothe invention.

In certain embodiments, the invention provides a method for producing amixture of antibodies to a target, the method comprising i) bringing anantibody display library comprising antibodies or antibody fragmentsinto contact with material comprising a target, ii) at least one step ofselecting antibodies or antibody fragments binding to the target, iii)identifying at least two antibodies or antibody fragments binding to thetarget, wherein at least two antibodies or antibody fragments comprise acommon light chain, iv) introducing a nucleic acid sequence encoding thelight chain and a nucleic acid sequence or nucleic acid sequencesencoding the heavy chains of at least two antibodies into a host cell,v) culturing a clone of the host cell under conditions conducive toexpression of nucleic acid sequences. The antibody display library maybe a phage display library, a ribosome display library, an mRNA displaylibrary, or a yeast display library. Steps i) and ii) may optionally berepeated one or more times.

The nucleic acid sequences encoding the antibodies obtained by the phagedisplay, ribosome display or yeast display method may be converted toencode any desired antibody format such as IgG1, IgG2, IgG3, IgG4, IgA,IgM, IgD, IgE, before introducing them into a host cell, using standardmolecular cloning methods and means known to one of ordinary skill inthe art (e.g., described in Boel et al., 2000, the entirety of which isincorporated herein by reference).

It will be clear to one of ordinary skill in the art that libraries inwhich only one light chain is represented are especially useful in lightof the invention, since all antibodies that can be obtained from such alibrary will have a common light chain that is functional in bindingtarget antigen with each of the heavy chains. In other words, inaccordance with the methods of the invention, the formation ofnon-functional light chain-heavy chain dimers is avoided. Phage antibodydisplay libraries having extensive H chain repertoires and unique orvery few L chain sequences have been disclosed in the art (Nissim etal., 1994; Vaughan et al., 1996, the entirety of which are incorporatedherein by reference). In general, the specificity of an antibody appearsto be determined to a large extent by its heavy chain. It is evenpossible to screen for and identify light chains that do not contributesignificantly to binding of the antibody, which light chains also couldbe suitably used according to the invention. It may also be possible tofollow the teachings of the invention but use one heavy chain and varythe light chains. However, the use of a common light chain and differentheavy chains appears preferable and the following observations supportthe idea that the specificity of an antibody appears to be dominated byits heavy chain sequence. In the process of receptor editing, amechanism of B-cells to monitor if their immunoglobulin receptor encodesa potentially harmful auto-antibody, B-cells expressing an auto-antibodyreplace the expressed heavy chain with another heavy chain whileretaining the expressed light chain. Thus, a new antibody specificity isgenerated that does not encode an auto-antibody. This shows that asingle light chain can successfully dimerize with multiple heavy chainsto form different antibody specificities (Nemazee, 2000; Casellas etal., 2001, the entirety of which are incorporated herein by reference).Series of transfected cell lines using a single heavy chain gene withdifferent light chain genes have been reported, the antibodies producedto a large extent maintaining their specificity, regardless of the lightchain (Radic et al., 1991, the entirety of which is incorporated hereinby reference).

Different antibodies have been obtained from a library that has beenconstructed using a single light chain (Nissim et al., 1994). Severalantibodies have been obtained from the library described by De Kruif etal. (1995, the entirety of which is incorporated herein by reference),which was constructed using seven light chains, that have the same lightchain but different specificities (see, e.g., Example 1: antibodiesbinding to EpCAM and to CD46, described in WO 01/48485 and WO 02/18948,respectively, the entirety of which are incorporated herein byreference).

Besides screening a phage library against a target, it will also bepossible to start with an antibody that has already proven its meritsand use the light chain of this antibody in the preparation of a libraryof heavy chains combined with this particular light chain only,according to methods known to one of ordinary skill in the art, such asphage display. Using this strategy, a monoclonal antibody can be used toobtain a mixture of antibodies according to the invention, functionallyresembling a polyclonal or oligoclonal antibody to the same target.Alternatively, a method reminiscent of the method described by Jesperset al. (1994, the entirety of which is incorporated herein by reference)to obtain a human antibody based on a functional rodent antibody can beused. The heavy chain of a known antibody of non-human origin is firstcloned and paired as a template chain with a repertoire of human lightchains for use in phage display, after which the phages are selected forbinding to the antigen or mixture of antigens. The selected light chainis, in turn, paired with a repertoire of human heavy chains displayed ona phage and the phages are selected again to find several heavy chainsthat, when paired with the light chain, are able to bind to the antigenor mixture of antigens of interest. This enables creating a mixture ofhuman antibodies against a target for which thus far only a non-humanmonoclonal antibody is described. It is possible that a mixtureaccording to the invention already has beneficial functional effectswhen the individual antibodies do not have high affinities for thetarget, whereas high affinities are often required for monoclonalantibodies to be effective. This would have the advantage that affinitymaturation may be required in less instances for methods and mixturesaccording to the invention than when an approach with monoclonalantibodies is envisaged.

The heavy and light chain coding sequences can be introducedsimultaneously or consecutively into the host cell. It is also an aspectof the invention to prepare a host cell comprising a recombinant nucleicacid encoding a light chain of an antibody. Such a cell can, forinstance, be obtained by transfection of the nucleic acid and,optionally, a clone can be identified that has a high expression of thelight chain. An established clone may then be used to add geneticinformation encoding 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, heavy chainsof the invention by introducing the nucleic acid molecules encodingthese into cells of the clone that already contains the light chain. Thenucleic acid molecules encoding the heavy chains may be introduced intothe host cell concomitantly. It is, of course, also possible tointroduce them consecutively, for instance, by using different selectionmarkers, which can be advantageous if not all heavy chains can beintroduced simultaneously because the cells do not take up enough copiesof recombinant nucleic acid molecules. Methods to introduce recombinantnucleic acid molecules into host cells are well known to one of ordinaryskill in the art and include transfection, electroporation, calciumphosphate precipitation, virus infection, and the like. One of ordinaryskill in the art has several possibilities to introduce more vectorswith nucleic acid sequences of interest into the same host cell, see,e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A LaboratoryManual, 2^(nd) edition, 1989; Current Protocols in Molecular Biology,Ausubel F. M., et al., eds, 1987; the series Methods in Enzymology(Academic Press, Inc.), the entirety of which are incorporated herein byreference.

Suitable dominant selection markers for introducing nucleic acids intoeukaryotic host cells, as used herein, may be G418 or neomycin(geneticin), hygromycin or mycophenolic acid, puromycin, and the like,for which genes encoding resistance are available on expression vectors.Further possibilities include, for instance, the use of vectorscontaining DHFR genes or glutamate synthetase to select in the presenceof methotrexate in a DHFR cell or the absence of glutamine in aglutamine auxotroph, respectively. The use of expression vectors withdifferent selection markers enables subsequent transfections with heavychain sequences of interest into the host cell, which already stablycontains other heavy chains introduced previously by use of otherselection markers. It is also possible to use selection markers that canbe used more than once, for instance, when containing mutations,introns, or weakened promoters that render them concentration-dependent(e.g., EP0724639; WO 01/32901; U.S. Pat. No. 5,733,779, the entirety ofwhich are incorporated herein by reference). Alternatively, a selectionmarker may be re-used by deleting it from the host cell after use, forexample, by site-specific recombination. A selectable marker locatedbetween sequences recognized by a site-specific recombinase, forexample, lox-sites or FRT-sites, is used for the generation of the firststable transfectant (for Cre-lox site-specific recombination, see,Wilson and Kola, 2001, the entirety of which is incorporated herein byreference). Subsequently, the selectable marker is excised from the hostcell DNA by the matching site-specific recombinase, for example, Cre orFlp. A subsequent transfection can suitably use the same selectionmarker.

Different host cell clones each comprising the genetic informationencoding a different light chain may be prepared. If the antibodies areidentified by an antibody display method, it is thus possible to prepareseveral host cells, each comprising one light chain present in theantibody display library. After identifying antibodies that bind to atarget using antibody display, the nucleic acid molecules encoding theheavy chains can be introduced into the host cell containing the commonlight chain that is capable of pairing to the heavy chains. It is,therefore, an aspect of the invention to provide a method for making ahost cell for production of a mixture of antibodies, the methodcomprising the steps of: introducing into the host cell a nucleic acidsequence encoding a light chain and nucleic acid sequence or sequencesencoding 3, 4, 5, 6, 7, 8, 9, 10, or more, different heavy chains thatare capable of pairing with the light chain, wherein the nucleic acidmolecules are introduced consecutively or simultaneously. It is, ofcourse, also possible to introduce at least two of the nucleic acidmolecules simultaneously, and introduce at least one other of thenucleic acid molecules consecutively.

In yet another aspect of the invention, a method is provided for makinga recombinant host cell for production of a mixture of antibodies, themethod comprising the step of: introducing a nucleic acid sequence ornucleic acid sequences encoding 2, 3, 4, 5, 6, 7, 8, 9, 10, or more,different heavy chains into a recombinant host cell comprising a nucleicacid sequence encoding a light chain capable of pairing with at leasttwo of the heavy chains.

If it appears that a recombinant host cell of the invention does notexpress sufficient light chain to dimerize with all of the expressed atleast two heavy chains, extra copies of the nucleic acid moleculesencoding the light chain may be transfected into the cell.

Besides random integration after transfection, methods to integrate thetransgenes in predetermined positions of the genome resulting infavorable expression levels can also be used according to the invention.Such methods may, for instance, employ site-specific integration byhomologous recombination (see, e.g., WO 98/41645, the entirety of whichis incorporated herein by reference) or make use of site-specificrecombinases (Gorman and Bullock, 2000, the entirety of which isincorporated herein by reference).

It is yet another aspect of the invention to provide a transgenicnon-human mammal or a transgenic plant comprising a nucleic acidsequence encoding a light chain and a nucleic acid sequence or nucleicacid sequences encoding at least two different heavy chains that arecapable of pairing with the light chain, wherein the nucleic acidsequences encoding the light and heavy chains are under the control of atissue-specific promoter. Promoters in plants may also be non-tissuespecific and general gene-expression elements, such as the CaMV 35Spromoter and nopaline synthase polyA addition site, can also be used.The light chain is a common light chain according to the invention. Inspecific embodiments, the transgenic animal or plant according to theinvention comprises 3, 4, 5, 6, 7, 8, 9, 10, or more, heavy chainsequences. Besides cell culture as a production system for recombinantproteins, the art also discloses the use of transgenic animals,transgenic plants and, for instance, transgenic chickens to produceproteins in the eggs, and the like to produce recombinant proteins ofinterest (Pollock et al., 1999; Larrick and Thomas, 2001; WO 91/08216,the entirety of which are incorporated herein by reference). Theseusually comprise the recombinant gene or genes encoding one or moreproteins of interest in operable association with a tissue-specificpromoter. It has, for instance, been shown that recombinant antibodiescan be produced at high levels in the milk of transgenic animals thatcontain the nucleic acids encoding a heavy and a light chain behind amammary gland-specific promoter (e.g., Pollock et al., 1999; WO95/17085, the entirety of which are incorporated herein by reference).Particularly useful in this respect are cows, sheep, goats, pigs,rabbits, mice, and the like, which can be milked to obtain antibodies.Useful promoters are the casein promoters, such as the β-caseinpromoter, the αS1-casein promoter, the whey acidic protein (WAP)promoter, the β-lactoglobulin promoter, the α-lactalbumin promoter, andthe like. Production of biopharmaceutical proteins in the milk oftransgenic mammals has been extensively described (e.g., Pollock et al.,1999, the entirety of which is incorporated herein by reference).Besides mammary gland-specific promoters, other tissue-specificpromoters may be used, directing the expression to the blood, urine,saliva, and the like. The generation of transgenic animals comprisingrecombinant nucleic acid molecules has been extensively documented andmay include micro-injection of oocytes (see, e.g., Wilmut and Clark,1991, the entirety of which is incorporated herein by reference),nuclear transfer after transfection (e.g., Schnieke et al., 1997, theentirety of which is incorporated herein by reference), infection byrecombinant viruses (e.g., U.S. Pat. No. 6,291,740, the entirety ofwhich is incorporated herein by reference), and the like. Nucleartransfer and cloning methods for mammalian cells are known to one ofordinary skill in the art, and are, for example, described in Campbellet al., 1996; Wilmut et al., 1997; Dinnyes et al., 2002; WO 95/17500;and WO 98/39416, the entirety of which are incorporated herein byreference. It is possible to clone animals and to generate lines ofanimals that are genetically identical, which renders it possible for aperson skilled in the art to create such a line once an individualanimal producing the desired mixture of antibodies has been identified.Alternatively, classical breeding methods can be used to generatetransgenic offspring. Strategies for the generation of transgenicanimals for production of recombinant proteins in milk are described inBrink et al., 2000, the entirety of which is incorporated herein byreference.

Transgenic plants or plant cells producing antibodies have also beendescribed (Hiatt et al., 1989; Peeters et al., 2001, the entirety ofwhich are incorporated herein by reference) and useful plants for thispurpose include corn, maize, tobacco, soybean, alfalfa, rice, and thelike. Constitutive promoters that can, for instance, be used in plantcells are the CaMV 35S and 19S promoters and Agrobacterium promoters nosand ocs. Other useful promoters are light-inducible promoters such asrbcS. Tissue-specific promoters can, for instance, be seed-specific,such as promoters from zein, napin, beta-phaseolin, ubiquitin, ortuber-specific, leaf-specific (e.g., useful in tobacco), root-specific,and the like. It is also possible to transform the plastid organelle byhomologous recombination to express proteins in plants.

Methods and means for expression of proteins in recombinant plants orparts thereof, or recombinant plant cell culture, are known to one ofordinary skill in the art and have been, for instance, described inGiddings et al., 2000; WO 01/64929; WO 97/42313; U.S. Pat. Nos.5,888,789, 6,080,560 (for practical guidelines, see Methods In MolecularBiology vol. 49 “Plant Gene Transfer And Expression Protocols,” H.Jones, 1995), the entirety of which are incorporated herein byreference. Other transgenic systems for producing recombinant proteinshave also been described, including the use of transgenic birds toproduce recombinant proteins in eggs (e.g., WO 97/47739, the entirety ofwhich is incorporated herein by reference) and the use of transgenicfish (e.g., WO 98/15627, the entirety of which is incorporated herein byreference), and can be used in combination with the teachings of theinvention to obtain mixtures of antibodies. It is also possible to usean in vitro transcription/translation or in vitro translation system forthe expression of mixtures of antibodies according to the invention. Itwill be clear to one of ordinary skill in the art that the teachings ofthe current invention will allow producing mixtures of antibodies insystems where recombinant nucleic acids encoding the light chain andheavy chains can be introduced and expressed. Preferably, such systemsare able to produce antibodies encoded by nucleic acid sequences,without the use of amplification of nucleic acid sequences in thesystems. In another aspect of the invention, a cell from a transgenicnon-human animal or a transgenic plant according to the invention isprovided. Such cells can be used to generate the animals or plantsaccording to the invention, using techniques known to one of ordinaryskill in the art, such as nuclear transfer or other known methods ofcloning whole organisms from single cells. The cells according to theinvention may also be obtained by introducing the light and at least twoheavy chain sequences into isolated cells of non-human animals orplants, which cells are capable of becoming part of a transgenic animalor plant. Particularly useful for such purposes are embryonic stemcells. These can contribute to the germ line and, therefore, the geneticinformation introduced into such cells can be passed to futuregenerations. In addition, plant cell cultures of cotton, corn, tomato,soybean, potato, petunia, and tobacco can be utilized as hosts whentransformed with the nucleic acid molecules encoding the light chain andthe heavy chains, for instance, by use of the plant-transformingbacterium A. tumefaciens or by particle bombardment or by infecting withrecombinant plant viruses.

In certain embodiments, the invention provides a pharmaceuticalcomposition comprising a mixture of recombinantly produced antibodiesand a suitable carrier, wherein at least two different heavy chains arerepresented in the mixture of recombinantly produced antibodies.Pharmaceutically acceptable carriers as used herein are exemplified, butnot limited to, adjuvants, solid carriers, water, buffers, or othercarriers used in the art to hold therapeutic components, or combinationsthereof. In particular embodiments, 3, 4, 5, 6, 7, 8, 9, 10, or more,different heavy chains are represented in the mixture. The mixture canbe obtained by mixing recombinantly produced monoclonal antibodies, butmay also be obtained by methods according to the invention. The mixturemay, therefore, comprise a common light chain for the antibodies. Themixture may comprise bispecific antibodies. The mixture may be producedfrom a clone that was derived from a single host cell, e.g., from apopulation of cells containing the same recombinant nucleic acidmolecules. The term “recombinantly produced” as used herein refers toproduction by host cells that produce antibodies encoded by recombinantnucleic acids introduced in such host cells or ancestors thereof. Itdoes not, therefore, include the classical method of producingpolyclonal antibodies, whereby a subject is immunized with an antigen orantigen-comprising mixture, after which the antibodies produced by thissubject are recovered from the subject, for example, from the blood.

In certain embodiments, the invention provides a mixture of antibodieswherein at least two heavy chains are represented for use in thetreatment or diagnosis of a human or animal subject. In another aspect,the invention provides the use of a mixture of antibodies wherein atleast two different heavy chains are represented for the preparation ofa medicament for use in the treatment or diagnosis of a disease ordisorder in a human or animal subject. In particular embodiments, 2, 3,4, 5, 6, 7, 8, 9, 10, or more, heavy chains are represented in themixture. The mixtures of antibodies may be mixtures of antibodiesaccording to the invention or obtained by methods according to theinvention. Antibodies present in the mixture may preferably comprise acommon light chain. The mixtures may comprise bispecific antibodies andmay be recombinantly produced from a clone that was derived from asingle host cell, i.e., from a population of cells containing the samerecombinant nucleic acid molecules. The targets may be used to screen anantibody display library, as described supra, to obtain 2, 3, 4, 5, 6,7, 8, 9, 10, or more, antibodies comprising a common light chain thatbind to the target and produce a mixture of these according to theteachings of the invention. Virtually any area of medicine wheremonoclonal antibodies can be used is amenable for the use of themixtures according to the invention. This can, e.g., include treatmentof auto-immune diseases and cancer, including solid tumors of the brain,head, neck, breast, prostate, colon, lung, and the like, as well ashematologic tumors, such as B-cell tumors. Neoplastic disorders whichcan be treated with the mixtures according to the invention includeleukemias, lymphomas, sarcomas, carcinomas, neural cell tumors, squamouscell carcinomas, germ cell tumors, metastases, undifferentiated tumors,seminomas, melanomas, myelomas, neuroblastomas, mixed cell tumors,neoplasias caused by infectious agents, and other malignancies. Targetsfor the antibody mixtures may include, but are not limited to, theHER-2/Neu receptor, other growth factor receptors (such as VEGFR1 andVEGFR2 receptors), B-cell markers (such as CD19, CD20, CD22, CD37, CD72,etc.), T-cell markers (such as CD3, CD25, etc.), other leukocyte cellsurface markers (such as CD33 or HLA-DR, etc.), cytokines (such as TNF),interleukins, receptors for these cytokines (such as members of the TNFreceptor family), and the like. It is anticipated that the use of suchmixtures of antibodies in the treatment of cancerous tissues or othercomplex multi-antigen-comprising cells such as microorganisms or viruseswill give rise to less occurrence of epitope-loss escape variants thanthe use of single monoclonal antibodies. Several treatments nowadays usepolyclonal mixtures of antibodies, which are derived from immunizedhumans or animals. These treatments may be replaced by use of themixtures according to the invention. Use of these mixtures can alsoinclude use in graft-versus-host rejections known in the art oftransplantation, e.g., by use of anti-thymocyte antibodies. It isanticipated that the mixtures of antibodies are superior to monoclonalantibodies in the treatment of complex antigens or antigen-comprisingmixtures such as bacteria or viruses. Therefore, use according to theinvention can also include use against strains of bacteria and fungi,e.g., in the treatment of infectious diseases due to pathogenic bacteriasuch as multidrug-resistant S. aureus and the like, fungi such asCandida albicans and Aspergillus species, yeast and the like. Themixtures according to the invention may also be used for post exposureprophylaxis against viruses, such as members of the genus Lyssavirus,e.g., rabies virus, or for therapeutic or prophylactic use againstviruses such as Varicella-Zoster Virus, Adenoviruses, RespiratorySyncitium Virus, Human Immunodeficiency Virus, Human Metapneumovirus,Influenzavirus, West Nile Virus, the virus causing Severe AcuteRespiratory Syndrome (SARS), and the like. Mixtures according to theinventions can also be used to protect against agents, both bacteria andviruses, and against toxic substances that are potential threats ofbiological warfare. Therefore, use according to the invention can alsoinclude use against strains of bacteria such as Bacillus anthracis,Clostridium botulinum toxin, Clostridium perfringens epsilon toxinYersinia Pestis, Francisella tulariensis, Coxiella burnetii, Brucellaspecies, Staphylococcus enterotoxin B, or against viruses such asVariola major, alphaviruses causing meningoencephalitis syndromes (EEEV,VEEV, and WEEV), viruses known to cause hemorrhagic fevers such asEbola, Marburg and Junin virus or against viruses such as Nipah virus,Hantaviruses, Tickborne encephalitis virus and Yellow fever virus oragainst toxins, for example, Ricin toxin from Ricinus communis and thelike. Use of the mixtures according to the invention can also includeuse against unicellular or multicellular parasites. Recombinant mixturesof antibodies according to the invention may become a safe alternativeto polyclonal antibodies obtained from pools of human sera for passiveimmunization or from sera of hyper-immunized animals. The mixtures maybe more efficacious than recombinant monoclonal antibodies in varioustherapeutic applications, including cancer, allergy, viral diseases,chronic inflammation, and the like.

It has been described that homodimerization of tumor-reactive monoclonalantibodies markedly increases their ability to induce growth arrest orapoptosis of tumor cells (Ghetie et al., 1997, the entirety of which isincorporated herein by reference). Possibly, when antibodies againstreceptors or other surface antigens on target cells, such as tumor cellsor infectious microorganisms, are produced according to the invention,the bispecific antibodies present in mixtures according to the inventionmay also cross-link different receptors or other antigens on the surfaceof target cells and, therefore, such mixtures may be very suitable forkilling such cells. Alternatively, when bispecific antibodies are lessdesirable, the invention also provides methods to recombinantly producemixtures of antibodies comprising mainly monospecific antibodies. It hasbeen described that the efficacy of treatment with Rituximab™ (anti-CD20monoclonal antibody) was increased when anti-CD59 antibodies were added(Herjunpaa et al., 2000, the entirety of which is incorporated herein byreference).

Therefore, it is expected that inclusion of antibodies against CD59 in amixture according to the invention comprising anti-tumor antibodies inthe form of B-cell receptor-recognizing antibodies increases thesensitivity of tumor cells to complement attack. It has also been shownthat a triple combination cocktail of anti-CD19, anti-CD22, andanti-CD38-saporin immunotoxins is much more effective than theindividual components in the treatment of human B-cell lymphoma in animmunodeficient mouse model (Flavell et al., 1997, the entirety of whichis incorporated herein by reference). Many other combinations may alsobe feasible and can be designed by one of ordinary skill in the art. Ingeneral, the use of antibody mixtures that are capable of recognizingmultiple B-cell epitopes will likely decrease the occurrence of escapevariants.

Another possible target is a transmembrane tyrosine kinase receptor,encoded by the Her-2/Neu (ErbB2) proto-oncogene (see, e.g., U.S. Pat.Nos. 5,772,997 and 5,783,186 for anti-Her2 antibodies, the entirety ofwhich are incorporated herein by reference). Her-2 is overexpressed on30% of highly malignant breast cancers and successful antibodies againstthis target marketed under the name HERCEPTIN™ (Trastuzumab) have beendeveloped. It has been shown that targeting multiple Her-2 epitopes witha mixture of monoclonal antibodies results in improved antigrowthactivity of a human breast cancer cell line in vitro and in vivo(Spiridon et al., 2002, the entirety of which is incorporated herein byreference). Her-2 may, therefore, be a good target for antibody mixturesaccording to the invention. Antibodies useful for this purpose can beobtained by methods described in the invention, including antibodydisplay methods.

Human antibodies are capable of eliciting effector function via bindingto immunoglobulin receptors on immune effector cells. Human IgG and, inparticular, IgG1 and IgG3, fix complement to induce CDC and interactwith Fcγ receptors to induce antibody-dependent cell-mediatedcytotoxicity (ADCC), phagocytosis, endocytosis, induction of respiratoryburst and release of inflammatory mediators and cytokines. Human IgAinteracts with FcαR, also resulting in efficient activation of ADCC andphagocytosis of target cells. Hence, due to the differentialdistribution of FcγR and FcαR on peripheral blood cells (Huls et al.,1999, the entirety of which is incorporated herein by reference), usinga mixture of antibodies directed against the target and consisting ofboth IgG and IgA would potentially maximize the recruitment andactivation of different immune effector cells. Such a mixture of bothIgG and IgA could be obtained by producing the IgG and IgA monoclonalantibody in a separate production process using two distinct productioncell lines, but could also be obtained from a single cell line producingboth the IgG and the IgA monoclonal antibody. This would have theadvantage that only a single production process has to be developed.Thus, when different heavy chains are mentioned, heavy chains differingin their constant regions are also encompassed in the invention. Theprinciple of using a common light chain can also be used for theproduction of a mixture of isotypes from a host cell. Therefore, certainembodiments of the invention provide a method for producing a mixture ofantibodies comprising different isotypes from a host cell, the methodcomprising the step of: culturing a host cell comprising a nucleic acidsequence encoding a light chain and nucleic acid sequences encoding atleast two heavy chains of different isotype that are capable of pairingwith the light chain, under conditions conducive to expression of thenucleic acid sequences. According to this aspect of the invention,different heavy chains may have identical variable regions and onlydiffer in their constant regions (i.e., be of different isotype and havethe same specificity). In a particular embodiment, the isotypes compriseat least an IgG and an IgA and/or IgM, preferably IgG1 or IgG3 and IgA.Other combinations of IgG1, IgG2, IgG3 and IgG4 can also be used. Inthese embodiments, bispecific antibodies will not be produced becausethe variable regions are the same.

In other embodiments of this aspect of the invention, not only theconstant regions of the heavy chains may differ, but also the variableregions, thereby giving rise to different specificities paired with thesame light chain. When bispecific antibodies are not desired for a givenpurpose, for example, because the mixtures of antibodies are lessefficacious because of the presence of the bispecific antibodies, it ispossible to use at least two heavy chains combined with the common lightchain according to the invention wherein the heavy chains differsufficient in their constant regions to reduce or prevent pairingbetween the different heavy chains, for example, by using heavy chainsof different isotypes, such as an IgG1 and an IgG3 (see FIG. 11 for aschematic representation). It is anticipated that the heavy chains ofdifferent isotype will pair much less efficient, if at all, compared tothe same heavy chains. Alternatively, it is also possible to engineerthe different heavy chains in their constant region such thathomodimerization is favored over heterodimerization, e.g., byintroducing self-complementary interactions (see, e.g., WO 98/50431 forpossibilities, such as “protuberance-into-cavity” strategies (see, WO96/27011, the entirety of which is incorporated herein by reference)).It is, therefore, another aspect of the invention to provide a methodfor producing a mixture of antibodies in a recombinant host, the methodincluding the step of: expressing in a recombinant host cell a nucleicacid sequence encoding a common light chain and nucleic acid sequencesencoding at least two different heavy chains that differ in the variableregion and that are capable of pairing with the common light chain, andwherein the heavy chains further differ in their constant regionssufficiently to reduce or prevent pairing between the different heavychains. In one embodiment, the heavy chains are of different isotype. Inspecific embodiments, 3, 4, 5, 6, 7, 8, 9, 10, or more, different heavychains are expressed. Mixtures of antibodies obtainable by this methodare also embodied in the invention. Such mixtures will comprise mainlymonospecific antibodies.

The teachings of the invention can also be used to obtain novelmultispecific antibodies or mixtures thereof. Therefore, in anotheraspect, the invention provides a method for producing a mixture ofantibodies comprising dimeric IgA isotype {(IgA)₂} antibodies in arecombinant host, wherein at least part of the dimeric IgA antibodieshave different binding regions in each of the IgA sub-units, the methodcomprising the step of: expressing in a recombinant host cell a nucleicacid sequence encoding a common light chain and nucleic acid sequencesencoding at least two different heavy chains of IgA isotype capable ofpairing to the common light chain, wherein the different heavy chainsdiffer in their variable region. Dimerization of the IgA molecules canbe enhanced by co-expressing J-chain (Yoo et al., 1999, the entirety ofwhich is incorporated herein by reference). Dimeric IgA antibodies havetwo specificities (see FIG. 9 for a schematic representation of onepossible form produced and present in the mixture).

In certain embodiments, the invention provides a method for producing amixture of antibodies comprising an IgM antibody having at least twodifferent specificities, the method comprising expressing in arecombinant host cell a nucleic acid sequence encoding a common lightchain and nucleic acid sequences encoding at least two different heavychains of IgM isotype, wherein the heavy chains are capable of pairingto the common light chain and form functional antigen-binding regions.Up to five specificities can be comprised in an IgM pentamer in thepresence of a J-chain and up to six in an IgM hexamer in the absence ofa J-chain (Yoo et al., 1999). Therefore, in specific embodiments, 3, 4,5, or 6 IgM heavy chains are co-expressed with the common light chainaccording to this aspect of the invention. See FIG. 10 for a schematicrepresentation of one of the possible forms that can be produced andpresent in the mixture according to this aspect of the invention, whenfive different heavy chains are expressed with a common light chain.Also provided is for IgA dimers, IgM pentamers or hexamers having atleast two different specificities. These molecules can be produced froma clone of a single host cell according to the invention. Such moleculesharboring antigen-binding regions with different specificities can binddifferent epitopes on the same antigen, different antigens on one cell,or different antigens on different cells, thereby cross-linking theantigens or cells.

In certain embodiments, the invention provides a method for identifyinga mixture of antibodies having a desired effect in a functional assay,the method comprising i) adding a mixture of antibodies in a functionalassay, and ii) determining the effect of the mixture in the assay,wherein the mixture of antibodies comprises antibodies having a commonlight chain. In a preferred embodiment, the mixture is comprised in acomposition of the invention.

Also provided is a method for recombinant expression of one or moreproteins in a single host cell, wherein at least four differentpolypeptides are expressed in the single host cell. Each polypeptide isindependently expressed and may be under control of a heterologouspromoter. The protein or proteins may be isolated separately or as amixture from a culture of the host cell. Preferably, the host cell ofthis embodiment is a human cell and/or may be derived from a retinacell, more preferably a cell comprising adenovirus E1 sequences in itsgenome, most preferably a PER.C6® cell (human retina cells that expressadenovirus E1A and E1B proteins).

EXAMPLES

The following examples are provided to illustrate the invention and arenot to be construed in any way to limit the scope of the invention. Thepractice of this invention will employ, unless otherwise indicated,conventional techniques of immunology, molecular biology, microbiology,cell biology, and recombinant DNA, which are within the skill of theart. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: ALaboratory Manual, 2^(nd) edition, 1989; Current Protocols in MolecularBiology, F. M. Ausubel, et al., eds, 1987; the series Methods inEnzymology (Academic Press, Inc.); PCR2: A Practical Approach, M. J.MacPherson, B. D. Hams, G. R. Taylor, eds, 1995; Antibodies: ALaboratory Manual, Harlow and Lane, eds, 1988, the entirety of which areincorporated herein by reference.

Example 1 Production of a Mixture of Monoclonal Antibodies with a CommonLight Chain and Two Different Heavy Chain-Variable Regions in a SingleCell

Clone UBS-54 and Clone K53 were previously isolated by selections on thecolorectal cell line SW40 (Huls et al., 1999) and on a heterogeneousmixture of mononuclear cells of a patient with multiple myeloma (WO02/18948, the entirety of which is incorporated herein by reference),respectively, with a semi-synthetic library (de Kruif et al., 1995b).Further studies revealed that clone UBS-54 and K53 bound to the EP-CAMhomotypic adhesion molecule (Huls et al., 1999) and the membranecofactor protein CD46 (WO 02/18948), respectively. DNA sequencing of theclones revealed that they were unique in the Heavy chain CDRs, but thatthey contained an identical light chain sequence (FIG. 3). The V_(H) andV_(L) of clones UBS-54 and K53 were inserted into an expression vectorcontaining the HAVT20 leader sequence and all the coding sequences forthe constant domains of a human IgG1 with a Kappa light chain by amethod essentially as described (Boel et al., 2000), which resulted inplasmids pUBS3000Neo and pCD46_(—)3000(Neo) (FIG. 4). These plasmidswere transiently expressed, either alone or in combination in PER.C6®cells (human retina cells that express adenovirus E1A and E1B proteins).In brief, each 80 cm² flask was transfected by incubation for four hourswith 140 μl lipofectamine+10 μg DNA (either pUBS3000Neo,pCD46_(—)3000(Neo) or 10 μg of both) in serum-free DMEM medium at 37° C.After four hours this was replaced with DMEM+10% FBS and the cells weregrown overnight at 37° C. Cells were then washed with PBS and the mediumwas replaced with Excell 525 medium (JRH Bioscience). The cells wereallowed to grow at 37° C. for six days, after which the cell culturesupernatant was harvested. Human IgG-specific ELISA analysis (describedin WO 00/63403, the entirety of which is incorporated herein byreference) indicated that IgG was present at approximately 10 μg/ml forall flasks containing expression plasmids. No IgG1 was present in acontrol flask which was not transfected with expression plasmid.

Human IgG from each supernatant was subsequently purified using ProteinA-affinity chromatography (Hightrap Protein A HP, cat. no. 1-040203)according to standard procedures, following recommendations of themanufacturer (Amersham Biosciences). After elution, samples wereconcentrated in a Microcon YM30 concentrator (Amicon) and bufferexchanged to 10 mM sodium phosphate, pH 6.7. Twelve μg of purified IgGwas subsequently analyzed on Isoelectric-focusing gels (Serva Pre-castIEF gels, pH range 3-10, cat. no. 42866). The samples were loaded on thelow pH side and after focusing, stained with colloidal blue (FIG. 5).Lane 1 shows transiently expressed K53, Lane 2 shows transientlyexpressed UBS-54 and Lane 3 shows the IgG sample of the cells in whichboth antibodies were co-transfected. Clearly, K53 and UBS-54 each have aunique pI profile and the sample from the co-transfection showed otherunique isoforms, with the major isoform having a pI in between those ofK53 and UBS-54. This is also anticipated on the basis of the theoreticpI when calculated with the ProtParam tool provided on the Expasyhomepage (expasy.ch; Appel et al., 1994, the entirety of which isincorporated herein by reference). K53 and UBS-54 have a theoretic pI of8.24 and 7.65, respectively, whereas an isoform representing aheterodimer of one UBS-54 heavy chain and one K53 heavy chain has atheoretical pI of 8.01. Assembly of such a heterodimer can only occurwhen a single cell translates both the heavy chain of K53 and the heavychain of UBS-54 and assembles these into a full length IgG moleculetogether with the common light chain.

Therefore, this experiment shows that it is possible to express twounique human IgG molecules in a single cell and that a heterodimerconsisting of these two unique binding specificities is also efficientlyformed.

Example 2 Production of a Mixture of Antibodies Against Human B-CellMarkers in a PER.C6® Cell Line (Human Retina Cells that ExpressAdenovirus E1A and E1B Proteins)-Derived Clone

A method for producing a mixture of antibodies according to theinvention, using expression in a recombinant host cell of a single lightchain and three different heavy chains capable of pairing to the singlelight chain to form functional antibodies, is exemplified herein and isschematically shown in FIG. 6. Phages encoding antibodies capable ofbinding proteins present on human B-cells, i.e., CD22, CD72 and MajorHistocompatibility Complex (MHC) class II (further referred to asHLA-DR) were previously isolated from a semi-synthetic phage library (deKruif et al., 1995; van der Vuurst de Vries & Logtenberg, 1999, theentirety of which is incorporated herein by reference). DNA sequencingof the V_(H) and V_(L) sequences of the phages clone B28 (anti-CD22),clone 1-2 (anti-HLA-DR) and clone II-2 (anti-CD72) revealed that theyall contain a unique V_(H) sequence but a common light chain sequence(Vλ3) with an identical CDR region (FIG. 7).

The V_(H) and V_(L) sequences of clones B28, I-1 and II-2 are clonedbehind the HAVT20 leader sequences of an expression plasmid comprising aheavy chain. An example of such a plasmid is pCRU-K01 (contains kappaheavy chain sequences that can be easily interchanged for lambda heavychain sequences if desired by a person skilled in the art), as depositedat the ECACC under number 03041601. The cloning gives rise to plasmidsencoding a full length human IgG1 with binding specificities for CD22,CD72 and HLA-DR. These plasmids will further be referred to aspCRU-CD22, pCRU-CD72 and pCRU-HLA-DR, respectively.

Stable PER.C6® (human retina cells that express adenovirus E1A and E1Bproteins)-derived cell lines are generated, according to methods knownto one of ordinary skill in the art (see, e.g., WO 00/63403), the celllines expressing antibodies encoded by genetic information on eitherpCRU-CD22, pCRU-CD72 or pCRU-HLA-DR and a cell line expressingantibodies encoded by all three plasmids. Therefore, PER.C6® cells(human retina cells that express adenovirus E1A and E1B proteins) areseeded in DMEM plus 10% FBS in tissue culture dishes (10 cm diameter) orT80 flasks with approximately 2.5×10⁶ cells per dish and kept overnightunder their normal culture conditions (10% CO₂ concentration and 37°C.). The next day, transfections are performed in separate dishes at 37°C. using Lipofectamine (Invitrogen Life Technologies) according tostandard protocols provided by the manufacturer, with either 1-2 μgpCRU-CD22, 1-2 μg pCRU-CD72, 1-2 μg pCRU-HLA-DR or 1 μg of a mixture ofpCRU-CD22, pCRU-CD72 and pCRU-HLA-DR. As a control for transfectionefficiency, a few dishes are transfected with a LacZ control vector,while a few dishes will not be transfected and serve as negativecontrols.

After four to five hours, cells are washed twice with DMEM and givenfresh medium without selection. The next day, the medium is replacedwith fresh medium containing 500 μg/ml G418. Cells are refreshed everytwo or three days with medium containing the same concentrations ofG418. About 20 to 22 days after seeding, a large number of colonies arevisible and from each transfection, at least 300 are picked and grownvia 96-well plates and/or 24-well plates via 6-well plates to T25flasks. At this stage, cells are frozen (at least one, but usually fourvials per sub-cultured colony) and production levels of recombinanthuman IgG antibody are determined in the supernatant using an ELISAspecific for human IgG1 (described in WO 00/63403). Also, at this stage,G418 is removed from the culture medium and never re-applied again. Fora representative number of colonies, larger volumes will be cultured topurify the recombinant human IgG1 fraction from the conditionedsupernatant using Protein A affinity chromatography according tostandard procedures. Purified human IgG1 from the various clones isanalyzed on SDS-PAGE, Iso-electric focusing (IEF) and binding to thetargets CD22, CD72 and HLA-DR using cell transfectants expressing thesehuman antigens on their cell surface (transfectants expressing CD72 andHLA-DR have been described by van der Vuurst-de Vries and Logtenberg,1999; a CD22 transfectant has been prepared according to similarstandard procedures in PER.C6® (human retina cells that expressadenovirus E1A and E1B proteins)).

Colonies obtained from the co-transfection with pCRU-CD22, pCRU-CD72 andpCRU-HLA-DR are screened by PCR on genomic DNA for the presence orabsence of each of the three constructs. The identity of the PCRproducts is further confirmed by DNA sequencing.

Next, it is demonstrated that a clonal cell line accounts for theproduction of each of the three binding specificities, i.e., provingthat a single cell is able to produce a mixture of more than twofunctional human IgGs. Therefore, a limited number of colonies, whichscreened positive for the production of each of the three bindingspecificities (both by PCR at the DNA level as well as in the specifiedbinding assays against CD22, CD72 and HLA-DR), are subjected to singlecell sorting using a fluorescence-activated cell sorter (FACS) (Becton &Dickinson FACS VANTAGE SE™ (high-performance, high-speed cell sorter)).Alternatively, colonies are seeded at 0.3 cells/well to guarantee clonaloutgrowth. Clonal cell populations, hereafter designated as sub-clones,are refreshed once a week with fresh medium. Sub-clones are grown andtransferred from 96-well plates via 24- and 6-well plates to T25 flasks.At this stage, sub-clones are frozen (at least one, but usually fourvials per sub-clone) and production levels of recombinant human IgG1antibody are determined in the supernatant using a human IgG1-specificELISA. For a representative number of sub-clones, larger volumes arecultured to purify the recombinant human IgG1 fraction from theconditioned supernatant using Protein A-affinity chromatographyaccording to standard procedures.

Purified human IgG1 from the various sub-clones is subsequently analyzedas described above for human IgG1 obtained from the parental clones,i.e., by SDS-PAGE, Iso-electric focusing (IEF) and binding to thetargets CD22, CD72 and HLA-DR. Sub-clones will also be screened by PCRon genomic DNA for the presence or absence of each of the threeconstructs pCRU-CD22, pCRU-CD72 and pCRU-HLA-DR. The identity of the PCRproducts is further confirmed by DNA sequencing.

Other methods such as Southern blot and/or FISH can also be used todetermine whether each of the three constructs are present in the clonalcell line.

Sub-clones that are proven to be transgenic for each of the threeconstructs are brought into culture for an extensive period to determinewhether the presence of the transgenes is stable and whether expressionof the antibody mixture remains the same, not only in terms ofexpression levels, but also for the ratio between the various antibodyisoforms that are secreted from the cell. Therefore, the sub-cloneculture is maintained for at least 25 population doubling times, eitheras an adherent culture or as a suspension culture. At every four to sixpopulation doublings, a specific production test is performed using thehuman IgG-specific ELISA and larger volumes are cultured to obtain thecell pellet and the supernatant. The cell pellet is used to assess thepresence of the three constructs in the genomic DNA, either via PCR,Southern blot and/or FISH. The supernatant is used to purify therecombinant human IgG1 fraction as described supra. Purified human IgG1obtained at the various population doublings is analyzed as described,i.e., by SDS-PAGE, Iso-electric focusing (IEF) and binding to thetargets CD22, CD72 and HLA-DR using cell transfectants expressing theseantigens.

Example 3 Screening of Clones Expressing Multiple Human IgGs for theMost Potent Mixture of Functional Human IgGs

Functionality of the antibody mixture is analyzed in cell-based assaysto determine whether the human IgG1 mixture inhibits proliferationand/or induces apoptosis of B-cell lines, such as, for example, Ramos.Other cell lines can also be used. In addition, the antibody mixturesare analyzed for their potential to induce antibody-dependent cellulartoxicity and complement-dependent cytotoxicity of, for example, Ramoscells.

In each of the following experiments, the functionality of the antibodymixture recognizing the targets CD22, CD72 and HLA-DR is analyzed andcan be compared to each of the individual IgG1 antibodies and to anequimolar combination of the three individual IgG1 specificities.

To assess the ability of the antibody mixtures to inhibit theproliferation of Ramos cells, these cells are incubated in 96-wellplates (0.1-1.0×10⁵/ml) with several concentrations (5-20 μg/ml) of theantibody mixtures against CD22, CD72 and HLA-DR for 24 hours. Theproliferation of the cells is measured by ³H-thymidine incorporationduring another 16 hours of culture. Inhibition of growth is determinedby plotting the percentage of ³H-thymidine incorporation compared tountreated cells (taken as 100% reference value).

To analyze apoptosis induction of Ramos cells, these cells arestimulated in 48-well plates (0.2-1.0×10⁶/ml) with severalconcentrations (5-20 μg/ml) of the antibody mixtures against the targetsCD22, CD72 and HLA-DR for 24 or 48 hours. After the incubation period,the phosphatidyl serine exposure on apoptotic cells is analyzed (G.Koopman et al., 1994, the entirety of which is incorporated herein byreference). Therefore, the cells are harvested, washed twice with PBSand are incubated at RT for 10 minutes with 100 μl FITC-labeled annexinV (Caltag) diluted 1:25 in annexin V-binding buffer (Caltag). Prior tothe analysis of the samples by flow cytometry (FACSCalibur, BectonDickinson, San Jose, Calif.), propidium iodide (PI)(Sigma) is added to afinal concentration of 5 μg/ml to distinguish necrotic cells (annexinV−/PI+) from apoptotic cells (annexin V+/PI−, early apoptotic cells;annexin V+/PI+, late apoptotic cells).

In an alternative assay, apoptosis is induced by cross-linking theantibody mixtures against CD22, CD72 and HLA-DR on the cell surface ofRamos cells with 25 μg/ml of F(ab)2 of goat-anti-human (Fc-specific)polyclonal antibodies (Jackson ImmunoResearch Laboratories, West Grove,Pa.) during the incubation period.

In another alternative assay, apoptosis is induced by incubating theRamos cells with several concentrations (5-20 μg/ml) of the antibodymixtures against CD22, CD72 and HLA-DR while co-incubating them with thechemosensitizing agents doxorubicin (Calbiochem) or dexamethasone (UMCU,Utrecht, NL).

Antibody-Dependent Cellular Cytotoxicity (ADCC) of the antibody mixturesis analyzed using peripheral blood mononuclear cells as effector cellsin a standard ⁵¹Cr release assay (Huls et al., 1999). To this purpose,1-3×10⁶ Ramos cells are labeled with 100 μCi (Amersham, Buckinghamshire,UK) for one hour at 37° C. After three washes with medium, the Ramostarget cells are plated in U bottom 96-well plates at 5×10³ cells/well.Peripheral blood mononuclear cells that are obtained from healthy donorsby Ficoll-Hypaque density gradients are then added to each well ateffector:target ratios ranging from 80:1 to 10:1 in triplicate. Thecells are incubated at 37° C. in the presence of various concentrationsof the antibody mixtures (5-20 μg/ml) in a final volume of 200 μl.

After four hours of incubation, part of the supernatant is harvested and⁵¹Cr release is measured. The percentage of specific lysis is calculatedusing the following formula: % specific lysis=([experimentalcpm−spontaneous cpm]/[maximal cpm−spontaneous cpm]×100%). Maximal ⁵¹Crrelease is determined by adding triton X-100 to a final concentration of1% to the target cells and spontaneous release is determined afterincubation of the target cells with medium alone.

Complement-dependent cytotoxicity is determined in a similar assay.Instead of the effector cells, now 50 μl human serum is added to thetarget cells. Subsequently, the assay is performed in the same manner.

Alternatively, ADCC and CDC of the antibody mixtures is determined usinga Europium release assay (Patel and Boyd, 1995, the entirety of which isincorporated herein by reference) or using an LDH release assay (Shieldset al., 2001, the entirety of which is incorporated herein byreference).

Example 4 Use of Phage Display to Isolate Multiple Phages with anIdentical V_(L) Sequence Against a Predefined Target (Her-2) andProduction in a Recombinant Host Cell of a Mixture of Antibodies Capableof Binding this Target

Phages displaying scFv fragments capable of binding multiple epitopespresent on the same protein, for example, the epidermal growth factorreceptor Her-2, can be isolated from a semi-synthetic phage library (deKruif et al., 1995a, b). It is possible to identify several of suchphages and select the ones comprising the same light chain sequence forfurther use according to the invention. The semi-synthetic library isformed by mixing seven sub-libraries that each contain a different lightchain (de Kruif et al., 1995a, b). It is, therefore, particularlypractical to use such a sub-library, containing only one light chain andmany heavy chains, for screening so that multiple antibodies with anidentical V_(L) sequence are obtained and further used for expressingthe antibody mixtures according to the invention.

For the selection of phages against Her-2, several fusion proteins aregenerated comprising different parts of the extracellular domain ofHer-2 that are fused to the CH2 and CH3 domains of human IgG1. For thispurpose, a pcDNA3.1zeo-expression vector (InVitrogen) has beenconstructed that contains in its multiple cloning region an XhoIrestriction site in the hinge region in frame prior to the CH2 and CH3domains of human IgG1. Using a Her-2 cDNA clone as a template, PCRfragments are generated using standard molecular biology techniquesknown to a person skilled in the art. These fragments consist of aunique 5′ restriction site, a start codon followed by a eukaryoticleader sequence that is linked in frame to either the totalextracellular (EC) domain of Her-2 or to a part of the EC domain ofHer-2 that is followed in frame by an XhoI restriction site. These PCRfragments are subsequently cloned in frame with the CH2-CH3 IgG1 regioninto the pcDNA3.1zeo-expression vector. In addition to the fusionprotein containing the total EC domain of Her-2, several smaller fusionproteins are generated containing non-overlapping fragments of the Her-2EC domain. These constructs encoding the Her-2-Ig fusion proteins areused for transient transfection of 293T cells using the lipofectaminereagent (Gibco). Five days after transfection, the supernatants of the293T cells are harvested and Her-2-Ig fusion proteins are purified usingprotein A-affinity chromatography according to standard procedures.

Her-2-Ig fusion proteins containing non-overlapping fragments of theHer-2 EC domain are coated for two hours at 37° C. onto the surface ofMAXISORP™ (polystyrene based modified surface with a high affinity forpolar groups) plastic tubes (Nunc) at a saturating concentration (0.5-5μg/ml). The tubes are blocked for one hour in 2% fat-free milk powderdissolved in PBS (MPBS). Simultaneously, 500 μl (approximately 10¹³ cfu)of a semi-synthetic phage display library (a sub-library according tothe terminology used above) in which only one Vκc light chain isrepresented (prepared as described by De Kruif et al. (1995a, b) andreferenced therein), is added to two volumes of 4% MPBS. In addition,human serum is added to a final concentration of 15% and blocking isallowed to proceed for 30 to 60 minutes. The Her-2-Ig-coated tubes areemptied and the blocked phage library is added. The tube is sealed androtated slowly for one hour, followed by two hours of incubation withoutrotation. The tubes are emptied and washed ten times in PBS containing0.1% Tween-20, followed by washing five times in PBS. One mlglycine-HCL, 0.05 M, pH 2.2 is added, and the tube is rotated slowly forten minutes. The eluted phages are added to 500 μl 1 M Tris-HCl pH 7.4.To this mixture, 3.5 ml of exponentially growing XL-1 blue bacterialculture is added. The tubes are incubated for 30 minutes at 37° C.without shaking. Subsequently, the bacteria are plated on 2TY agarplates containing ampicillin, tetracycline and glucose. After overnightincubation of the plates at 37° C., the colonies are scraped from theplates and used to prepare an enriched phage library, essentially asdescribed by De Kruif et al. (1995a). Briefly, scraped bacteria are usedto inoculate 2TY medium containing ampicillin, tetracycline and glucoseand are grown at 37° C. to an OD_(600nm) of ˜0.3. Helper phages areadded and allowed to infect the bacteria after which the medium ischanged to 2TY containing ampicillin, tetracycline and kanamycin.Incubation is continued overnight at 30° C. The next day, the bacteriaare removed from the 2TY medium by centrifugation, after which thephages are precipitated using polyethylene glycol 6000/NaCl. Finally,the phages are dissolved in a small volume of PBS-1% BSA,filter-sterilized and used for a next round of selection. Theselection/re-infection procedure is performed twice. After the secondround of selection, individual E. coli colonies are used to preparemonoclonal phage antibodies. Essentially, individual colonies are grownto log phase and infected with helper phages, after which phage antibodyproduction is allowed to proceed overnight. Phage antibody containingsupernatants are tested in ELISA for binding activity to Her-2-totalEC-Ig coated 96-well plates.

Selected phage antibodies that are obtained in the screen describedabove are validated by ELISA for specificity. For this purpose, Her-2-Igfusion proteins containing non-overlapping fragments of the Her-2 ECdomain are coated to Maxisorp ELISA plates. After coating, the platesare blocked in 2% MPBS. The selected phage antibodies are incubated inan equal volume of 4% MPBS. The plates are emptied, washed once in PBS,after which the blocked phages are added. Incubation is allowed toproceed for one hour, the plates are washed in PBS 0.1% Tween-20 andbound phages are detected using an anti-M13 antibody conjugated toperoxidase. The procedure is performed simultaneously using a controlphage antibody directed against thyroglobulin (De Kruif et al. 1995a,b), which serves as a negative control.

In another assay, the selected phage antibodies are analyzed for theirability to bind BT474 human breast cancer cells that express Her-2. Forflow cytometry analysis, phage antibodies are first blocked in an equalvolume of 4% MPBS for 15 minutes at 4° C. prior to the staining of theBT474 cells. The binding of the phage antibodies to the cells isvisualized using a biotinylated anti-M13 antibody (Santa CruzBiotechnology) followed by streptavidin-phycoerythrin (Caltag).

Alternatively, phage antibodies recognizing multiple epitopes on Her-2are selected using a method based upon competition of phage binding toHer-2 with binding of the well-characterized murine anti-Her-2antibodies HER50, HER66 and HER70 (Spiridon et al., 2002, the entiretyof which is incorporated herein by reference). To this purpose, 2×10⁶BT474 cells are incubated at 4° C. with approximately 10¹³ cfu (0.5 ml)of a semi-synthetic phage display library in which only one Vκ1 lightchain is represented, prepared as described supra, and blocked with twovolumes of medium containing 10% of FBS. The mixture is slowly rotatedat 4° C. for two hours in a sealed tube.

Subsequently, non-bound phages are removed by two washes with 50 ml ofcold medium containing 10% FBS. Hereafter, phages recognizing multipleepitopes on Her-2 are eluted by resuspending the BT474 cells in 1 ml ofcold medium containing saturating concentrations (5-20 μg/ml) of theHER50, HER66 and HER70 murine anti-Her-2 antibodies. The cells are lefton ice for 10 minutes, spun down and the supernatant containing theanti-Her-2 phage antibodies is used to reinfect XL1-Blue cells asdescribed supra.

From the panel of Her-2-specific phage antibodies generated by thescreens described above, three phage antibodies are selected thatrecognize three different non-overlapping epitopes on the Her-2 protein.

The V_(H) sequences and the unique Vκ1 light chain sequence of theseclones, provisionally designated Vκ1HER2-1, Vκ1HER2-2 and Vκ1HER2-3, arecloned behind the HAVT20 leader sequences of expression plasmid pCRU-K01(ECACC deposit 03041601), or a similar expression plasmid, to obtainplasmids encoding a full-length human IgG1-K with binding specificitiesfor Her-2. These plasmids are provisionally designated aspCRU-Vκ1HER2-1, pCRU-Vκ1HER2-2 and pCRU-Vκ1HER2-3, respectively.

Stable PER.C6® (human retina cells that express adenovirus E1A and E1Bproteins)-derived cell lines are generated, according to methods knownto one of ordinary skill in the art, the cell lines expressingantibodies encoded by genetic information on either pCRU-Vκ1HER2-1,pCRU-Vκ1HER2-2 or pCRU-Vκ1HER2-3 and a cell line expressing antibodiesencoded by all three plasmids. Therefore, PER.C6® cells are seeded inDMEM plus 10% FBS in tissue culture dishes (10 cm diameter) or T80flasks with approximately 2.5×10⁶ cells per dish and kept overnightunder their normal culture conditions (10% CO₂ concentration and 37°C.). The next day, transfections are performed in separate dishes at 37°C. using Lipofectamine (Invitrogen Life Technologies) according tostandard protocols provided by the manufacturer, with either 1-2 μgpCRU-Vκ1HER2-1, 1-2 μg pCRU-Vκ1HER2-2, 1-2 μg pCRU-Vκ1HER2-3 or 1 μg ofa mixture of pCRU-Vκ1HER2-1, pCRU-Vκ1HER2-2 and pCRU-Vκ1HER2-3. As acontrol for transfection efficiency, a few dishes are transfected with aLacZ control vector, while a few dishes are not transfected and serve asnegative controls.

After five hours, cells are washed twice with DMEM and re-fed with freshmedium without selection. The next day, medium is replaced with freshmedium containing 500 μg/ml G418. Cells are refreshed every two or threedays with medium containing the same concentrations of G418. About 20 to22 days after seeding, a large number of colonies are visible and fromeach transfection, at least 300 are picked and grown via 96-well platesand/or 24-well plates via 6-well plates to T25 flasks. At this stage,cells are frozen (at least one, but usually four vials per sub-culturedcolony) and production levels of recombinant human IgG antibody aredetermined in the supernatant using an ELISA specific for human IgG1.Also, at this stage, G418 is removed from the culture medium and neverre-applied again. For a representative number of colonies, largervolumes are cultured to purify the recombinant human IgG1 fraction fromthe conditioned supernatant using Protein A-affinity chromatographyaccording to standard procedures. Purified human IgG1 from the variousclones is analyzed on SDS-PAGE, Iso-electric focusing (IEF), assayedbinding to Her-2-Ig fusion proteins by ELISA, and analyzed for bindingto Her-2 on the surface of BT474 cells by flow cytometry.

Clones obtained from the co-transfection of pCRU-Vκ1HER2-1,pCRU-Vκ1HER2-2 and pCRU-Vκ1HER2-3 are screened by PCR on genomic DNA forthe presence or absence of each of the three constructs. The identity ofthe PCR products is further confirmed by DNA sequencing.

Next, it is demonstrated that a clonal cell line accounts for theproduction of each of the three binding specificities. Therefore, alimited number of colonies, which screened positive for the productionof each of the three binding specificities (both by PCR at the DNA levelas well as in the specified binding assays against Her-2), are subjectedto single cell sorting using a fluorescence-activated cell sorter (FACS)(Becton & Dickinson FACS VANTAGE SE™). Alternatively, colonies areseeded at 0.3 cells/well to guarantee clonal outgrowth.

Clonal cell populations, hereafter designated as sub-clones, arerefreshed once a week with fresh medium. Sub-clones are grown andtransferred from 96-well plates via 24- and 6-well plates to T25 flasks.At this stage, sub-clones are frozen (at least one, but usually fourvials per sub-clone) and production levels of recombinant human IgG1antibody are determined in the supernatant using a human IgG1-specificELISA. For a representative number of sub-clones, larger volumes arecultured to purify the recombinant human IgG1 fraction from theconditioned supernatant using Protein A-affinity chromatographyaccording to standard procedures.

Purified human IgG1 from the various sub-clones is subsequently analyzedas described above for human IgG1 obtained from the parental clones,i.e., by SDS-PAGE, Iso-electric focusing (IEF) and binding to Her-2.Sub-clones will also be screened by PCR on genomic DNA for the presenceor absence of each of the three constructs pCRU-Vκ1HER2-1,pCRU-Vκ1HER2-2 and pCRU-Vκ1HER2-3. The identity of the PCR products isfurther confirmed by DNA sequencing.

Other methods such as Southern blot and/or FISH can also be used todetermine whether each of the three constructs are present in the clonalcell line.

Sub-clones that are proven to be transgenic for each of the threeconstructs are brought into culture for an extensive period to determinewhether the presence of the transgenes is stable and whether expressionof the antibody mixture remains the same, not only in terms ofexpression levels, but also for the ratio between the various antibodiesthat are secreted from the cell. Therefore, the sub-clone culture ismaintained for at least 25 population doubling times, either as anadherent culture or as a suspension culture. At every four to sixpopulation doublings, a specific production test is performed using thehuman IgG-specific ELISA and larger volumes are cultured to obtain thecell pellet and the supernatant. The cell pellet is used to assess thepresence of the three constructs in the genomic DNA, either via PCR,Southern blot and/or FISH. The supernatant is used to purify therecombinant human IgG1 fraction as described supra. Purified human IgG1obtained at the various population doublings is analyzed as described,i.e., by SDS-PAGE, Iso-electric focusing (IEF) and binding to Her-2 byELISA and by flow cytometry using BT474 cells.

Functionality of the antibody mixture of anti-Her-2 antibodies isanalyzed in cell-based assays to determine whether the human IgG1mixture inhibits proliferation and/or induces apoptosis of BT474 cells.In addition, the antibody mixtures are analyzed for their potential toinduce antibody-dependent cellular toxicity and complement-dependentcytotoxicity of BT474 cells.

In each of the experiments described below, the functionality of theantibody mixture recognizing Her-2 can be analyzed and compared to eachof the individual IgG1 antibodies and to an equimolar combination of thethree individual monospecific IgG1 molecules.

To assess the ability of the antibody mixtures to inhibit theproliferation of BT474 cells, these cells are allowed to adhereovernight in 96-well plates (1.5×10⁵/well) and are subsequentlyincubated with several concentrations (5-20 μg/ml) of the antibodymixtures against Her-2 for 72 hours. The proliferation of the cells ismeasured by ³H-thymidine incorporation during the last six hours ofculture. Inhibition of growth is determined by plotting the percentageof ³H-thymidine incorporation compared with untreated cells (taken as100% reference value).

To analyze apoptosis induction of BT474 cells, these cells are allowedto adhere overnight in 48-well plates (2.5×10⁵/well in 1 ml) and aresubsequently incubated with several concentrations (5-20 μg/ml) of theantibody mixtures against Her-2 for four hours. Hereafter, the cells areharvested by trypsinization, washed twice with PBS and incubated at RTfor ten minutes with 100 μl FITC-labeled annexin V (Caltag) diluted 1:25in annexin V-binding buffer (Caltag). Prior to the analysis of thesamples by flow cytometry (FACSCalibur, Becton Dickinson, San Jose,Calif.) propidium iodide (PI)(Sigma) is added to a final concentrationof 5 μg/ml to distinguish necrotic cells (annexin V⁻/PI⁺) from apoptoticcells (annexin V⁺/PI⁻, early apoptotic cells; annexin V⁺/PI⁺, lateapoptotic cells).

Antibody-Dependent Cellular Cytotoxicity of the antibody mixtures isanalyzed using peripheral blood mononuclear cells as effector cells andBT474 cells as target cells in a standard ⁵¹Cr release assay asdescribed supra (Huls et al., 1999). Complement-dependent cytotoxicityis determined in a similar assay. Instead of the effector cells, now 50μl human serum is added to the target cells. Subsequently, the assay isperformed as described supra.

Alternatively, ADCC and CDC of the antibody mixtures is determined usinga Europium release assay (Patel and Boyd, 1995) or using an LDH releaseassay (Shields et al., 2001).

The functionality of the antibody mixtures against Her-2 is also testedusing in vivo animal models, such as, for instance, described inSpiridon et al., 2002.

Example 5 Expression of Different Functional Human IgGs in the Milk ofTransgenic Animals

The V_(H) and V_(H) sequences of phages against proteins present onhuman B-cells, i.e., CD22 (clone B28), CD72 (clone II-2) and HLA-DR(clone I-2) (FIG. 7) are cloned into expression plasmid pBC1 (asprovided in the pBC1 Mouse Milk Expression System, Invitrogen LifeTechnologies) to obtain mammary gland- and lactation-specific expressionof these human IgG molecules in transgenic animals, according to themanufacturer's instructions. These mammary gland-specific expressionvectors encoding the antibody sequences for anti-CD22, anti-CD72 andanti-HLA-DR, are introduced into the murine germline according to themanufacturer's instructions. Obtained pups are screened for the presenceof each of the three constructs by PCR on DNA isolated from the tail.Pups, either male or female, confirmed for being transgenic for each ofthe three antibodies, are weaned and matured. Female transgenic mice arefertilized at the age of 6-8 weeks and milk samples are obtained atseveral time points after gestation. Male transgenic mice are mated withnon-transgenic females and female transgenic offspring (as determinedwith PCR as described above) is mated and milked as described above forthe female transgenic founders. Whenever needed, female or maletransgenic founders are mated for another generation to be able toobtain sufficient amounts of transgenic milk for each founder line.Transgenic milk is analyzed for the presence of human IgG with a humanIgG-specific ELISA, which does not cross-react with mouse IgG or othermouse milk components. Human IgG is purified from transgenic mouse milkusing Protein A-affinity chromatography according to standardprocedures. Purified human IgG is analyzed on SDS-PAGE, Iso-electricfocusing and binding on the targets CD22, CD72 and HLA-DR. Functionalityof the antibody mixture is analyzed as described supra.

Example 6 Production of an IgA/IgG Mixture Against a Predefined Targetin a PER.C6® (Human Retina Cells that Express Adenovirus E1A and E1BProteins)-Derived Clone

The V_(H)-V_(L) sequences of the phage UBS-54 directed against thehomotypic adhesion molecule EP-CAM (Huls et al., 1999) was not onlycloned into a vector encoding the constant domains of a human IgG1 withKappa light chain (expression vector pUBS3000Neo), but also into anexpression vector encoding the constant domains of a human IgA1 withKappa light chain (expression vector pUBS54-IgA, FIG. 8). Hence,antibodies derived from pUBS3000Neo and pUBS54-IgA do bind to the sameepitope on EPCAM. The only differences antibodies derived frompUBS3000Neo and pUBS54-IgA are in the sequences encoding the constantdomains of the heavy chain, resulting in either an IgG1 or IgA1 isotype.The Kappa light chain sequences of these two vectors are identical.

Stable PER.C6® (human retina cells that express adenovirus E1A and E1Bproteins)-derived cell lines expressing antibodies encoded by geneticinformation on pUBS3000Neo and pUBS54-IgA are generated by procedureswell known to persons skilled in the art. Therefore, PER.C6® cells(human retina cells that express adenovirus E1A and E1B proteins) areseeded in DMEM plus 10% FBS in tissue culture dishes (10 cm diameter) orT80 flasks with approximately 2.5×10⁶ cells per dish and kept overnightunder their normal culture conditions (10% CO₂ concentration and 37°C.). The next day, transfections are performed in separate dishes at 37°C. using Lipofectamine (Invitrogen Life Technologies) according tostandard protocols provided by the manufacturer, with either 1-2 μgpUBS3000Neo and pUBS54-IgA. As a control for transfection efficiency, afew dishes are transfected with a LacZ control vector, while a fewdishes are not transfected and serve as negative controls.

After four to five hours, cells are washed twice with DMEM and givenfresh medium without selection. The next day, medium is replaced withfresh medium containing 500 μg/ml G418. Cells are refreshed every two orthree days with medium containing the same concentrations of G418. About20 to 22 days after seeding, a large number of colonies are visible andfrom each transfection, at least 300 are picked and grown via 96-wellplates and/or 24-well plates via 6-well plates to T25 flasks. At thisstage, cells are frozen (at least one, but usually four vials persub-cultured colony) and production levels of recombinant human IgG andhuman IgA antibody are determined in the supernatant using an ELISAspecific for human IgG1 as well as an ELISA specific for human IgA.Also, at this stage, G418 is removed from the culture medium and neverre-applied again. For a representative number of colonies, largervolumes are cultured to purify the recombinant human IgG1 and human IgAfraction from the conditioned supernatant using, for instance, acombination of Protein L- or LA-affinity chromatography, cation exchangechromatography, hydrophobic interaction chromatography and gelfiltration. Purified human immunoglobulins from the various clones areanalyzed on SDS-PAGE, Iso-electric focusing (IEF) and binding to thetarget EPCAM using cell lines having a high expression of this molecule.The clones will also be screened by PCR on genomic DNA for the presenceor absence of pUBS3000Neo and pUBS54-IgA. The identity of the PCRproducts is further confirmed by DNA sequencing.

A limited number of clones, which are screened positive for theproduction of both EPCAM IgG1 and EPCAM IgA, are subjected to singlecell sorting using a fluorescence-activated cell sorter (FACS) (BectonDickinson FACS VANTAGE SE™). Alternatively, colonies are seeded at 0.3cells/well to guarantee clonal outgrowth. Clonal cell populations,hereafter designated as sub-clones, are refreshed once a week with freshmedium. Sub-clones are grown and transferred from 96-well plates via 24-and 6-well plates to T25 flasks. At this stage, sub-clones are frozen(at least one, but usually four vials per sub-clone) and productionlevels of recombinant human IgG1 and IgA antibody are determined in thesupernatant using a human IgG1-specific ELISA and a human IgA-specificELISA. For a representative number of sub-clones, larger volumes arecultured to purify the recombinant human IgG1 and human IgA1 fractionfrom the conditioned supernatant using, for instance, a combination ofProtein L- or LA-affinity chromatography, cation exchangechromatography, hydrophobic interaction chromatography and gelfiltration. Purified human immunoglobulins from the various clones areanalyzed on SDS-PAGE, Iso-electric focusing (IEF) and binding to thetarget EPCAM using cell lines having a high expression of this molecule.

Sub-clones will also be screened by PCR on genomic DNA for the presenceor absence of pUBS3000Neo and pUBS54-IgA. The identity of the PCRproducts is further confirmed by DNA sequencing.

Other methods such as Southern blot and/or FISH may also be used todetermine whether both constructs are present in the clonal cell line.

Example 7 Production of a Human IgG1/IgG3 Mixture Against MultipleTargets in a Clonal PER.C6® Cell Line (Human Retina Cells that ExpressAdenovirus E1A and E1B Proteins)

Phage clone UBS-54 and Clone K53 (FIG. 3) were obtained as described inExample 1. The V_(H) and V_(L) of clone UBS-54 was inserted into anexpression vector containing the HAVT20 leader sequence and all thecoding sequences for the constant domains of a human IgG1 with a Kappalight chain by a method essentially as described (Boel et al., 2000).The resulting plasmid was designated as pUBS3000Neo (FIG. 4). It will beclear that expression vectors containing heavy chain constant domains ofany desired isotype can be constructed by routine methods of molecularbiology, using the sequences of these regions that are all available inthe art. The V_(H) and V_(L) sequences of Phage clone K53 are clonedinto an expression vector containing the HAVT20 leader sequence and allthe coding sequences for the constant domains of a heavy chain of ahuman IgG3 with a Kappa light chain by a method essentially as described(Boel et al., 2000). This expression vector is designated as pK53IgG3.

These plasmids are transiently expressed, either alone or incombination, in PER.C6® cells (human retina cells that expressadenovirus E1A and E1B proteins). In brief, each 80 cm² flask istransfected by incubation for four hours with 140 μl lipofectamine+10 μgDNA (either pUBS3000Neo, pK53IgG3 or 10 μg of both) in serum-free DMEMmedium at 37° C. After four hours, this is replaced with DMEM+10% FBSand the cells are grown overnight at 37° C. Cells are then washed withPBS and the medium is replaced with Excell 525 medium (JRH Bioscience).The cells are allowed to grow at 37° C. for six days, after which thecell culture supernatant is harvested. Human IgG-specific ELISAanalysis, i.e., measuring all IgG sub-types, is done to determine theIgG concentration in transfected and non-transfected PER.C6® cells(human retina cells that express adenovirus E1A and E1B proteins). HumanIgG from each supernatant is subsequently purified using ProteinA-affinity chromatography (Hightrap Protein A HP, cat. no. 1-040203)according to standard procedures, following recommendations of themanufacturer (Amersham Biosciences). After elution, samples areconcentrated in a Microcon YM30 concentrator (Amicon) and bufferexchanged to 10 mM sodium phosphate, pH 6.7. Samples are analyzed forbinding to the targets EPCAM and CD46 using cell lines having a highexpression of these molecules such as LS174T cells. Twelve μg ofpurified IgG, either transiently expressed UBS-54 IgG1, K53 IgG3 or IgGfrom the cells in which both antibodies were co-transfected, issubsequently analyzed on iso-electric-focusing gels (Serva Pre-cast IEFgels, pH range 3-10, cat. no. 42866). Samples are loaded on the low pHside and, after focusing, stained with colloidal blue. The pI values ofthe major isoforms for each sample are determined to illustrate whetherthere has been expression of UBS-54 IgG1, K53 IgG3 or bispecificheterodimers, depending on how the cells were transfected. Theidentification of heterodimers would indicate that single cells havetranslated both the IgG3 heavy chain of K53 and the IgG1 heavy chain ofUBS-54 and assembled these into a full-length IgG molecule together withthe common light chain.

The absence of bispecific heterodimers indicates that it is possible totranslate both the IgG3 heavy chain of K53 and the IgG1 heavy chain ofUBS-54 in single cells, but that these do not assemble into afull-length IgG molecule together with the common light chain, i.e.,there is preferential binding of IgG1 and IgG3 heavy chains. This could,however, also be explained by the lack of co-expression of UBS-54 IgG1and K53 IgG3. Therefore, stable clonal cell lines expressing bothpUBS3000Neo and pK53IgG3 are generated by procedures as such well knownto persons skilled in the art. PER.C6® cells (human retina cells thatexpress adenovirus E1A and E1B proteins) are seeded in DMEM plus 10% FBSin tissue culture dishes (10 cm diameter) or T80 flasks withapproximately 2.5×10⁶ cells per dish and kept overnight under theirnormal culture conditions (10% CO₂ concentration and 37° C.). The nextday, transfections are performed in separate dishes at 37° C. usingLipofectamine (Invitrogen Life Technologies) according to standardprotocols provided by the manufacturer, with either 1-2 μg pUBS3000Neo,pK53IgG3 or both. As a control for transfection efficiency, a few dishesare transfected with a LacZ control vector, while a few dishes will benot transfected and serve as negative controls.

After four to five hours, cells are washed twice with DMEM and givenfresh medium without selection. The next day, medium is replaced withfresh medium containing 500 μg/ml G418. Cells are refreshed every two orthree days with medium containing the same concentrations of G418. About20 to 22 days after seeding, a large number of colonies are visible andfrom each transfection, at least 300 are picked and grown via 96-wellplates and/or 24-well plates via 6-well plates to T25 flasks. At thisstage, cells are frozen (at least one, but usually four vials persub-cultured colony) and production levels of recombinant human IgGantibody are determined in the supernatant using an ELISA specific forall sub-types of human IgG. Also, at this stage, G418 is removed fromthe culture medium and never re-applied again. For a representativenumber of colonies, larger volumes are cultured to purify therecombinant human IgG from the conditioned supernatant using ProteinA-affinity chromatography (Hightrap Protein A HP, cat. no. 1-040203)according to standard procedures, following recommendations of themanufacturer (Amersham Biosciences). Purified human immunoglobulins fromthe various clones are analyzed on SDS-PAGE, Iso-electric focusing (IEF)and binding to the targets EPCAM and CD46 using cell lines having a highexpression of these molecules such as LS174T cells. The clones are alsoscreened by PCR on genomic DNA for the presence or absence ofpUBS3000Neo and pK53IgG3. The identity of the PCR products is furtherconfirmed by DNA sequencing.

A limited number of clones, which are screened positive for theproduction of both EPCAM IgG1 and K53 IgG3, are subjected to single cellsorting using a fluorescence-activated cell sorter (FACS) (BectonDickinson FACS VANTAGE SE™). Alternatively, colonies are seeded at 0.3cells/well to guarantee clonal outgrowth. Clonal cell populations,hereafter designated as sub-clones, are refreshed once a week with freshmedium. Sub-clones are grown and transferred from 96-well plates via 24-and 6-well plates to T25 flasks. At this stage, sub-clones are frozen(at least one, but usually four vials per sub-clone) and productionlevels of recombinant human IgG antibody are determined in thesupernatant using a human IgG-specific ELISA. For a representativenumber of sub-clones, larger volumes are cultured to purify therecombinant human IgG fraction from the conditioned supernatant usingProtein A-affinity chromatography (Hightrap Protein A HP, cat. no.1-040203) according to standard procedures, following recommendations ofthe manufacturer (Amersham Biosciences). Purified human immunoglobulinsfrom the various clones are analyzed on SDS-PAGE, Iso-electric focusing(IEF) and binding to the targets EPCAM and CD46 using cell lines havinga high expression of this molecules, such as, for instance, LS174Tcells, or transfectants expressing these molecules.

Sub-clones are also screened by PCR on genomic DNA for the presence orabsence of pUBS3000Neo and pK53IgG3. The identity of the PCR products isfurther confirmed by DNA sequencing.

Other methods such as Southern blot and/or FISH may also be used todetermine whether both constructs are present in the clonal cell line.

Once the clonal sub-clones are available and confirmed positive for theexpression of both UBS-54 IgG1 and K53 IgG3, the presence of functionalK53 and UBS-54 shows that it is possible to generate a mixture offunctional IgGs with different isotypes with the common light chain in asingle cell. Analysis of the expression of bispecific antibodies bindingboth EpCAM and CD46 will reveal to what extent the different heavychains having a different sub-type will pair, which will influence theamount of bispecific antibodies produced. It is expected that no or verylow levels of bispecific antibodies will be found in this case.

Example 8 Selection of Phage Carrying Single Chain Fv FragmentsSpecifically Recognizing Rabies Virus Glyco Protein (RVGP) Using RVGP-IgFusion Protein, and Expression of Mixtures of Antibodies Against theRabies Virus

This example describes the production of mixtures of antibodies againstthe rabies virus as another potential target. As an antigen, the RabiesVirus Glycoprotein (RVGP) is chosen, but other rabies antigens may bechosen or included as well for this purpose. Several monoclonalantibodies recognizing RVGP have already been described in the art, andpolyclonal antibodies have been recognized to be useful in treatment ofrabies infections as well (e.g., EP0402029; EP0445625, the entirety ofwhich are incorporated herein by reference).

Antibody fragments are selected using antibody phage display librariesand MAbstract™ technology, essentially as described in U.S. Pat. No.6,265,150 and in WO 98/15833, the entirety of which is incorporatedherein by reference. All procedures are performed at room temperatureunless stated otherwise. The sequence of RVGP is available to one ofordinary skill in the art for cloning purposes (e.g., Yelverton et al.,1983, the entirety of which is incorporated herein by reference). AnRVGP-Ig fusion protein consisting of whole RVGP fused genetically to theCH2 and CH3 domains of human IgG1 is produced using vector pcDNA3.1Zeo-CH2-CH3 expressed in PER.C6® (human retina cells that expressadenovirus E1A and E1B proteins) and coated for two hours at 37° C. ontothe surface of MAXISORP™ (polystyrene based modified surface with a highaffinity for polar groups) plastic tubes (Nunc) at a concentration of1.25 μg/ml. The tubes are blocked for one hour in 2% fat-free milkpowder dissolved in PBS (MPBS). Simultaneously, 500 μl (approximately10¹³ cfu) of a phage display library expressing single chain Fvfragments (scFvs) essentially prepared as described by De Kruif et al.(1995a, b) and references therein, is added to two volumes of 4% MPBS.In this experiment, selections are performed using fractions of theoriginal library constructed using only one single variable light chaingene species (e.g., a “Vκ1”-library). In addition, human serum is addedto a final concentration of 15% and blocking is allowed to proceed for30 to 60 minutes. The RVGP-Ig-coated tubes are emptied and the blockedphage library is added. The tube is sealed and rotated slowly for onehour, followed by two hours of incubation without rotation. The tubesare emptied and washed ten times in PBS containing 0.1% Tween-20,followed by washing five times in PBS. One ml glycine-HCL, 0.05 M, pH2.2 is added, and the tube is rotated slowly for ten minutes. The elutedphages are added to 500 μl 1 M Tris-HCl pH 7.4. To this mixture, 3.5 mlof exponentially growing XL-1 blue bacterial culture is added. The tubesare incubated for 30 minutes at 37° C. without shaking. Then, thebacteria are plated on 2TY agar plates containing ampicillin,tetracycline and glucose. After overnight incubation of the plates at37° C., the colonies are scraped from the plates and used to prepare anenriched phage library, essentially as described by De Kruif et al.(1995a, b). Briefly, scraped bacteria are used to inoculate 2TY mediumcontaining ampicillin, tetracycline and glucose and grown at atemperature of 37° C. to an OD_(600nm) of ˜0.3. Helper phages are addedand allowed to infect the bacteria, after which the medium is changed to2TY containing ampicillin, tetracycline and kanamycin. Incubation iscontinued overnight at 30° C. The next day, the bacteria are removedfrom the 2TY medium by centrifugation, after which the phages areprecipitated using polyethylene glycol 6000/NaCl. Finally, the phagesare dissolved in a small volume of PBS-1% BSA, filter-sterilized andused for a next round of selection. The selection/re-infection procedureis performed twice.

After the second round of selection, individual E. coli colonies areused to prepare monoclonal phage antibodies. Essentially, individualcolonies are grown to log-phase and infected with helper phages, afterwhich phage antibody production is allowed to proceed overnight. Phageantibody-containing supernatants are tested in ELISA for bindingactivity to human RVGP-Ig coated 96-well plates.

Selected phage antibodies that are obtained in the screen describedabove are validated in ELISA for specificity. For this purpose, humanRVGP-Ig is coated to Maxisorp ELISA plates. After coating, the platesare blocked in 2% MPBS. The selected phage antibodies are incubated inan equal volume of 4% MPBS. The plates are emptied, washed once in PBS,after which the blocked phages are added. Incubation is allowed toproceed for one hour, the plates are washed in PBS 0.1% Tween-20 andbound phages are detected using an anti-M13 antibody conjugated toperoxidase. As a control, the procedure is performed simultaneouslyusing a control phage antibody directed against thyroglobulin (De Kruifet al. 1995a, b), which serves as a negative control.

The phage antibodies that bind to human RVGP-Ig are subsequently testedfor binding to human serum IgG to exclude the possibility that theyrecognized the Fc part of the fusion protein.

In another assay, the phage antibodies are analyzed for their ability tobind PER.C6® cells (human retina cells that express adenovirus E1A andE1B proteins) that express RVGP. To this purpose, PER.C6® cells (humanretina cells that express adenovirus E1A and E1B proteins) aretransfected with a plasmid carrying a cDNA sequence encoding RVGP orwith the empty vector and stable transfectants are selected usingstandard techniques known to a person skilled in the art (e.g., J. E.Coligan et al. (2001), Current Protocols In Protein Science, volume I,John Wiley & Sons, Inc. New York, the entirety of which is incorporatedherein by reference). For flow cytometry analysis, phage antibodies arefirst blocked in an equal volume of 4% MPBS for 15 minutes at 4° C.prior to the staining of the RVGP- and control-transfected PER.C6® cells(human retina cells that express adenovirus E1A and E1B proteins). Theblocked phages are added to a mixture of unlabeled control-transfectedPER.C6® cells (human retina cells that express adenovirus E1A and E1Bproteins) and RGVP-transfected PER.C6® cells that have been labeledgreen using a lipophylic dye (PKH67, Sigma). The binding of the phageantibodies to the cells is visualized using a biotinylated anti-M13antibody (Santa Cruz Biotechnology), followed bystreptavidin-phycoerythrin (Caltag). Anti RVGP scFv selectively stainsthe PER.C6® RVGP transfectant while they do not bind the controltransfectant.

An alternative way of screening for phages carrying single chain Fvfragments specifically recognizing human RVGP, is by use ofRVGP-transfected PER.C6® cells (human retina cells that expressadenovirus E1A and E1B proteins).

PER.C6® cells (human retina cells that express adenovirus E1A and E1Bproteins) expressing membrane-bound RVGP are produced as describedsupra. Phage selection experiments are performed as described supra,using these cells as target. A fraction of the phage library comprisedof scFv phage particles using only one single scFv species (500 μl,approximately 10¹³ cfu) is blocked with 2 ml RPMI/10% FCS/1% NHS for 15minutes at RT. Untransfected PER.C6® cells (human retina cells thatexpress adenovirus E1A and E1B proteins) (˜10×10⁶ cells) are added tothe PER.C6-RVGP cells (˜1.0×10⁶ cells). This mixture is added to theblocked light chain restricted phage library and incubated for 2.5 hourswhile slowly rotating at 4° C. Subsequently, the cells are washed twiceand were resuspended in 500 μl RPMI/10% FCS and incubated with a murineanti-RVGP antibody (Becton Dickinson) followed by a phycoerythrin(PE)-conjugated anti-mouse-IgG antibody (Caltag) for 15 minutes on ice.The cells are washed once and transferred to a 4 ml tube. Cell sortingis performed on a FACSvantage fluorescence-activated cell sorter (BectonDickinson) and RVGP (PE positive) cells are sorted. The sorted cells arespun down, the supernatant is saved and the bound phages are eluted fromthe cells by resuspending the cells in 500 μl 50 mM Glycin pH2.2followed by incubation for five minutes at room temperature. The mixtureis neutralized with 250 μl 1 M Tris-HCl pH 7.4 and added to the rescuedsupernatant. Collectively, these phages are used to prepare an enrichedphage library as described above. The selection/re-infection procedureis performed twice. After the second round of selection, monoclonalphage antibodies are prepared and tested for binding to RVGP-PER.C6®cells and untransfected PER.C6® cells (human retina cells that expressadenovirus E1A and E1B proteins) as described supra. Phages that arepositive on RVGP-transfected cells are subsequently tested for bindingto the RVGP-IgG fusion protein in ELISA as described supra.

The selected scFv fragments are cloned in a human IgG1 format, accordingto methods known in the art (e.g., Boel et al., 2000). To this purpose,the V_(L) fragment shared by the selected scFv is PCR amplified usingoligos that add appropriate restriction sites. A similar procedure isused for the V_(H) genes. Thus, modified genes are cloned in expressionpCRU-K01 (ECACC deposit 03041601), which results in expression vectorsencoding a complete huIgG1 heavy chain and a complete human light chaingene having the same specificity as the original phage clone. By thismethod, three different heavy chains are cloned into separate expressionvectors, while only one of the vectors needs to comprise the commonlight chain sequence. These expression vectors are provisionallydesignated pCRU-RVGP-1, pCU-RVGP-2, and pCRU-RVGP-3. Alternatively,these three vectors may lack DNA encoding the V_(L) region, which canthen be encoded in a fourth, separate expression vector not encoding aheavy chain. It is also possible to have V_(L) sequences present in allthree or two of the three vectors comprising the different V_(H)sequences.

Stable PER.C6® (human retina cells that express adenovirus E1A and E1Bproteins)-derived cell lines are generated, according to methods knownto one of ordinary skill in the art (see, e.g., WO 00/63403), the celllines expressing antibodies encoded by genetic information on eitherpCRU-RVGP-1, pCRU-RVGP-2 or pCRU-RVGP-3 and a cell line expressingantibodies encoded by all three plasmids. Therefore, PER.C6® cells areseeded in DMEM plus 10% FBS in tissue culture dishes (10 cm diameter) orT80 flasks with approximately 2.5×10⁶ cells per dish and kept overnightunder their normal culture conditions (10% CO₂ concentration and 37°C.). The next day, transfections are performed in separate dishes at 37°C. using Lipofectamine (Invitrogen Life Technologies) according tostandard protocols provided by the manufacturer, with either 1-2 μgpCRU-RVGP-1, 1-2 μg pCRU-RVGP-2, 1-2 μg pCRU-RVGP-3 or 1 μg of a mixtureof pCRU-RVGP-1, pCRU-RVGP-2 and pCRU-RVGP-3. As a control fortransfection efficiency, a few dishes are transfected with a LacZcontrol vector, while a few dishes will not be transfected and serve asnegative controls.

After four to five hours, cells are washed twice with DMEM and givenfresh medium without selection. The next day, the medium is replacedwith fresh medium containing 500 μg/ml G418. Cells are refreshed everytwo or three days with medium containing the same concentrations ofG418. About 20 to 22 days after seeding, a large number of colonies arevisible and from each transfection, at least 300 are picked and grownvia 96-well plates and/or 24-well plates via 6-well plates to T25flasks. At this stage, cells are frozen (at least one, but usually fourvials per sub-cultured colony) and production levels of recombinanthuman IgG antibody are determined in the supernatant using an ELISAspecific for human IgG1 (described in WO 00/63403). Also, at this stage,G418 is removed from the culture medium and never re-applied again. Fora representative number of colonies, larger volumes will be cultured topurify the recombinant human IgG1 fraction from the conditionedsupernatant using Protein A-affinity chromatography according tostandard procedures. Purified human IgG1 from the various clones isanalyzed on SDS-PAGE, Iso-electric focusing (IEF) and binding to thetarget RVGP using an RVGP PER.C6-transfectant described above.

Colonies obtained from the co-transfection with pCRU-RVGP-1, pCRU-RVGP-2and pCRU-RVGP-3 are screened by PCR on genomic DNA for the presence orabsence of each of the three constructs. The identity of the PCRproducts is further confirmed by DNA sequencing.

A limited number of colonies, which screened positive for the productionof each of the three binding specificities (both by PCR at the DNA levelas well as in the specified binding assays against RVGP), are subjectedto single cell sorting using a fluorescence-activated cell sorter (FACS)(Becton & Dickinson FACS VANTAGE SE™).

Alternatively, colonies are seeded at 0.3 cells/well to guarantee clonaloutgrowth. Clonal cell populations, hereafter designated as sub-clones,are refreshed once a week with fresh medium. Sub-clones are grown andtransferred from 96-well plates via 24- and 6-well plates to T25 flasks.At this stage, sub-clones are frozen (at least one, but usually fourvials per sub-clone) and production levels of recombinant human IgG1antibody are determined in the supernatant using a human IgG1-specificELISA. For a representative number of sub-clones, larger volumes arecultured to purify the recombinant human IgG1 fraction from theconditioned supernatant using Protein A-affinity chromatographyaccording to standard procedures.

Purified human IgG1 from the various sub-clones is subsequently analyzedas described above for human IgG1 obtained from the parental clones,i.e., by SDS-PAGE, Iso-electric focusing (IEF) and binding to the targetRVGP.

Sub-clones are also screened by PCR on genomic DNA for the presence orabsence of each of the three constructs pCRU-RVGP-1, pCRU-RVGP-2 andpCRU-RVGP-3. The identity of the PCR products is further confirmed byDNA sequencing.

Other methods such as Southern blot and/or FISH can also be used todetermine whether each of the three constructs are present in the clonalcell line.

Sub-clones that are proven to be transgenic for each of the threeconstructs are brought into culture for an extensive period to determinewhether the presence of the transgenes is stable and whether expressionof the antibody mixture remains the same, not only in terms ofexpression levels, but also for the ratio between the various antibodyisoforms that are secreted from the cell. Therefore, the sub-cloneculture is maintained for at least 25 population doubling times, eitheras an adherent culture or as a suspension culture. At every four to sixpopulation doublings, a specific production test is performed using thehuman IgG-specific ELISA and larger volumes are cultured to obtain thecell pellet and the supernatant. The cell pellet is used to assess thepresence of the three constructs in the genomic DNA, either via PCR,Southern blot and/or FISH. The supernatant is used to purify therecombinant human IgG1 fraction as described supra. Purified human IgG1obtained at the various population doublings is analyzed as described,i.e., by SDS-PAGE, Iso-electric focusing (IEF) and binding to the targetRVGP.

The efficacy of the antibody mixtures against rabies is tested in invitro cell culture assays where the decrease in spread of rabies virusis measured, as well as in in vivo animal models infected by rabies.Such models are known to one of ordinary skill in the art and are, e.g.,described in EP0402029.

Example 9 Production of a Mixture of Antibodies with a Common LightChain and Three Different Heavy Chain-Variable Regions in a Single Cell

A method for producing a mixture of antibodies according to theinvention using expression in a recombinant host cell of a single lightchain and three different heavy chains capable of pairing to the singlelight chain to form functional antibodies, is exemplified herein and isschematically shown in FIG. 6.

Human IgGs UBS54 and K53 against the EP-CAM homotypic adhesion molecule(Huls et al., 1999) and the membrane cofactor protein CD46 (WO02/18948), respectively, are described in Example 1. Another clone thatwas identified to bind to cofactor protein CD46 was clone 02-237(sequence of V_(H) provided in FIG. 12, SEQ ID NO:10). DNA sequencing ofthis clone revealed that it contained the same light chain as UBS54 andK53 but a unique heavy chain-variable sequence (see alignment in FIG.3). As a result, the CDR3 of the heavy chain of 02-237 differs at fourpositions from that of K53 (see alignment in FIG. 13). The heavy andlight chain-variable sequences of phage 02-237 were cloned into theexpression plasmid pCRU-K91 (pCRU-K01 is deposited at the EuropeanCollection of Cell Cultures (ECACC) under number 03041601), whichcontains the heavy and light chain constant domains for an IgG1antibody.

The resulting plasmid was designated pgG102-237. Due to the cloningstrategy followed, the resulting N-terminus of the light chain of 02-237as encoded by pgG102-237 differed slightly from the N-terminus of UBS54and K53 as present by pUBS3000Neo, pCD46_(—)3000(Neo), respectively(FIG. 3). Plasmid pgG102-237 was transiently produced in human 293(T)cells or stably in PER.C6® cells (human retina cells that expressadenovirus E1A and E1B proteins). It appeared that purified 02-237 IgGhad a much higher affinity for purified CD46 (FIG. 14) than K53 IgG,i.e., the affinity had increased from 9.1×10⁻⁷ M to 2.2×10⁻⁸ M for K53and 02-237, respectively. Also, 02-237 bound much better to CD46 onhuman coloncarcinoma LS174T cells than K53 (FIG. 15).

Stable PER.C6® (human retina cells that express adenovirus E1A and E1Bproteins)-derived cell lines expressing a combination of the plasmidspUBS3000Neo, pCD46_(—)3000(Neo) and pgG102-237 encoding human IgG 02-237were generated according to methods known as such to one of ordinaryskill in the art (see, e.g., WO 00/63403). Therefore, PER.C6® cells(human retina cells that express adenovirus E1A and E1B proteins) wereseeded in DMEM plus 10% FBS in tissue culture dishes (10 cm diameter)with approximately 2.5×10⁶ cells per dish and kept overnight under theirnormal culture conditions (10% CO₂ concentration and 37° C.). The nextday, transfections were performed in separate dishes at 37° C. usingLipofectamine (Invitrogen Life Technologies) according to standardprotocols provided by the manufacturer, with 2 μg of an equimolarmixture of pUBS3000Neo, pCD46_(—)3000(Neo) and pgG102-237. As negativecontrol for selection, a few dishes were not transfected.

After four to five hours, cells were washed twice with DMEM and givenfresh medium without selection. The next day, medium was replaced withfresh medium containing 500 μg/ml G418. Cells were refreshed every twoor three days with medium containing the same concentrations of G418.About 20 to 22 days after seeding, a large number of colonies werevisible and about 300 were picked and grown via 96-well plates and/or24-well plates via 6-well plates to T25 flasks. During sub-culturing,production levels of recombinant human IgG antibody were determined inthe supernatant using an ELISA specific for human IgG1 (described in WO00/63403). About 25% of all colonies appeared to be positive in thishighly specific assay. The production levels measured at this stage werecomparable to the levels when a single IgG is expressed in PER.C6® cells(human retina cells that express adenovirus E1A and E1B proteins)(expression of a single IgG described in Jones et al., 2003). It isimportant to stress that these high expression levels were obtainedwithout any methods for amplification of the transgene and that theyoccur at a low copy number of the transgene.

The 30 best producing colonies were frozen down in vials and the 19highest producing clones were selected for purification of the IgG(Table 1). They were sub-cultured in T80 flasks and human IgG from eachclone was subsequently purified using Protein A-affinity chromatography.Therefore, 15 to 25 ml of conditioned medium was loaded on a 5 mlProtein A FF Sepharose column (Amersham Biosciences). The column waswashed with 4 mM phosphate buffered saline, pH 7.4 (PBS) before elutionwith 0.1 M citrate pH 3.0. The eluted fraction was subsequently desaltedon a Sephadex G25 Fine HiPrep Desalting column (Amersham Biotech) toPBS. The concentration of the purified IgG fraction was determined byabsorbance measurement at 280 nm using a coefficient of 1.4 for a 0.1%(w/v) solution (Table 1).

The purified IgG samples were analyzed on non-reduced and reducedSDS-PAGE and IEF. Non-reduced SDS-PAGE (FIG. 16A) showed that all IgGsamples migrated comparable to the control K53 or 02-237 as anassembled, intact IgG molecule of approximately 150 kDa. On reducedSDS-PAGE (FIG. 16B), the IgG samples migrated as heavy and light chainsof about 50 and 25 kDa, respectively, comparable to the heavy and lightchain of the control K53 or 02-237.

On IEF, the purified IgG fractions were first compared to a mixture ofequal amounts of K53, UBS54 and 02-237 (FIG. 17). Clearly, some of thesamples contained isoforms with a unique pI profile when compared to themixture containing purified K53, UBS54 and 02-237. Some major uniqueisoforms have a pI in between the pI of K53 and 02-237 on one hand andUBS54 on the other hand. This is also anticipated on the basis of thetheoretic pI when calculated with the ProtParam tool provided on theExpasy homepage (expasy.ch; Appel et al., 1994). K53, 02-237 and UBS54have a theoretic pI of 8.24, 8.36 and 7.65, respectively, whereas anisoform representing a heterodimer of one UBS54 heavy chain and one K53heavy chain, has a theoretical pI of 8.01. Assembly of such aheterodimer can only occur when a single cell translates both the heavychain of K53 and the heavy chain of UBS54 and assembles these into afull-length IgG molecule together with the common light chain. Hence,these results suggest that certain clones at least express twofunctional antibodies. To confirm the unique identity of some of theisoforms, samples of the most interesting clones were run in parallelwith K53, UBS54 and 02-237, either alone or in a mixture (FIG. 18). Thisfurthermore showed that some clones expressed at least two antibodies(241, 282, 361). Moreover, it provided evidence that some clones expressall three functional antibodies (280 and 402).

To confirm that the clones expressed IgG mixtures comprising all threeheavy chains, peptide mapping (Garnick, 1992; Gelpí, 1995, the entiretyof which are incorporated herein by reference) was used to analyze thepolyclonal IgG fraction. We previously employed peptide mapping torecover 99% of the protein sequence of K53.

Based on the protein sequence provided in FIG. 12, the mass of thetheoretical tryptic peptides of K53, UBS54 and 02-237 was calculated(Table II and III). A few unique peptides for each IgG could beidentified, for instance, the CDR3 peptides for K53, 02-237 and UBS54with a Mw of 2116.05, 2057.99 and 2307.15 Da, respectively. Next, atryptic digest of Poly1-280 was prepared and this was analyzed usingLC-MS (FIG. 19).

Peptides with Mw of 2116, 2057 and 2308 Da, representing the unique CDR3peptides of K53, 02-237 and UBS54, respectively, were detected. Theprecise amino acid sequence of these peptides (as listed in Table III)was confirmed by MS-MS analysis (Tables IV, V and VI). The presence ofthe two unique N-terminal light chain peptides with Mw of 2580 and 2554Da, respectively, was also confirmed. The peptide mapping dataunequivocally showed that a mixture of antibodies comprising a commonlight chain and three different heavy chains was expressed by PER.C6®(human retina cells that express adenovirus E1A and E1B proteins) clonePoly1-280. Also, clones 055, 241 and 402 were screened by peptidemapping. Clones 241 and 402 were confirmed positive for all three heavychain sequences, whereas clone 055 only showed expression of the heavychains of K53 and 02-237, and not of UBS54. This confirms the IEFscreening (FIG. 18) where no UBS54-related band was seen in sample 055.

Poly1-280 was analyzed by BIACORE™ (surface plasmon resonance) forbinding to CD46 (FIG. 20). The affinity of poly1-280 for CD46 was2.1×10⁻⁸ M, which shows that the IgG mixture contains CD46-bindingmolecules having the same affinity as 02-237 IgG alone.

Taken together, this experiment shows that it is possible to express amixture of functional IgG molecules comprising three unique heavy chainsin a single cell and that next to the homodimers, heterodimersconsisting of two binding specificities are also formed. Furthermore,the frequency of clones expressing three different heavy chains suggeststhat it will also be possible to obtain clones expressing at least 4, 5,or more, heavy chains, using the same procedure. In the case where itwould be difficult to obtain clones expressing higher numbers of heavychains, a clone expressing at least three heavy chains according to theinvention can be used to introduce more heavy chains in a separate roundof transfection, for instance by using a different selection marker.

Next, it was demonstrated that a single cell is able to produce amixture of more than two functional human IgGs. Therefore, clones 241,280 and 402, which were screened positive for the production of each ofthe three IgGs, both by IEF and MS, were subjected to limiting dilution,i.e., seeded at 0.3 cells/well in 96-well plates to guarantee clonaloutgrowth.

Clonal cell populations, hereafter designated as sub-clones, wererefreshed once a week with fresh medium. Sub-clones were grown andtransferred from 96-well plates via 24- and 6-well plates, T25, T80 andT175 flasks. At the T80 stage, sub-clones were frozen. Production levelsof recombinant human IgG1 antibody were determined in the supernatantusing a human IgG1-specific ELISA. For each parental clone, threesub-clones were chosen and cultured in a few T175 flasks to obtainsufficient conditioned medium for purification using Protein A-affinitychromatography as described above.

Purified human IgG1 from the sub-clones was subsequently analyzed asdescribed above for human IgG1 obtained from the parental clone byiso-electric focusing (IEF). The result is shown in FIG. 21. Sub-clonesfrom clone poly 1-241 each have the same pattern, but differ from theparental clone in that they appear to miss certain bands.

Sub-clones from clone poly 1-280 all appear to differ from each otherand from the parental clone. Patterns obtained by IEF for sub-clonesfrom parental clone poly 1-402 are identical for all three sub-clonesand the parent clone.

From these data, it can be concluded that clone 402 is stably producinga mixture of antibodies. This demonstrates that it is feasible toproduce a mixture of antibodies according to the invention from a singlecell clone. The clones have undergone about 25 population doublings(cell divisions) from the transfection procedure up to the firstanalysis (shown in FIG. 18) under selection pressure and, from thatpoint on, have undergone about 30 population doublings during thesub-cloning procedure in the absence of selection pressure before thematerial analyzed in FIG. 21 was harvested. Therefore, the production ofa mixture of antibodies from a clone from a single cell can be stableover at least 30 generations.

Purified IgG1 from the parental 241, 280 and 402 clones, and sub-clones,were also analyzed for binding reactivity towards the CD46 and EpCAMantigens. To this end, cDNA of EpCAM, CD46, and control antigen CD38were cloned into expression vectors pcDNA (Invitrogen). These vectorswere transfected into CHO (dhfr-) cells using Fugene (Roche) accordingto the protocol supplied by the manufacturer. Cells were cultured inIscove's medium containing 10% FBS and HT supplement (Gibco). Afterculturing for two days, cells were harvested by trypsinization andsuspended in PBS-1% BSA (PBSB) for use in FACS analysis.

Purified IgG1 of the clones producing the mixtures of antibodies andcontrol IgG1 samples of anti-GBSIII, an anti-CD72 antibody (02-004), aswell as antibodies from anti-EpCAM clone UBS54 and anti-CD46 clones K53and 02-237, were diluted in PBSB to a concentration of 20 μg IgG1/ml.Twenty μl of each was added to 200,000 transfected cells and incubatedon ice for one hour. Thereafter, cells were washed once in ice-coldPBSB. Bound IgG was then detected using incubation with goat-anti-humanIgG-biotin followed by streptavidin-PE. After a final washing step,cells were suspended in PBSB containing 1 μg/ml propidium iodide. Thesamples were analyzed on a FACS (FACSvantage, Becton Dickinson). Livecells were gated and Mean Fluorescent Intensities (MFI) were calculatedfrom the FACS plots. The results are represented in FIG. 22. Asexpected, UBS54 bound selectively to EpCAM-transfected cells and 02-237and K53 bound selectively to CD46 transfectants, while unrelatedantibodies did not bind to these transfectants.

The results demonstrate that binding activities towards both EpCAM andCD46 were present in the purified IgG1 preps of most clones expressing amixture of antibodies according to the invention, demonstrating that amixture of functional antibodies was produced by sub-clones that haveundergone more than 30 cell divisions and that result from a singlecell. In sub-clone 280-015, binding patterns towards CD46 and EpCAM weresimilar as in the parent clone poly 1-280, in contrast to the otherclones.

It should be stated that the quantitative aspect of this assay is notcompletely clear. Routine screening, for example, by a functional test,can be used to find a clone with the desired expression profile.Quantitative aspects may also be included in such screens. Suchscreening allows for the identification of desired clones, which expressthe mixture of antibodies with a given functionality in a quantitativelystable manner.

All references, including publications, patents, and patentapplications, cited herein are hereby incorporated by reference to thesame extent as if each reference were individually and specificallyindicated to be incorporated by reference and were set forth in itsentirety herein.

TABLE I Overview of the clones used for purification of IgG. ScreeningPurification Clone ELISA Conc. in feed Purified Poly1- (μg/ml) (μg/ml)(mg) 209 6.1 98 1.37 233 10.0 53 0.75 234 8.0 51 0.71 241 6.6 91 1.42250 12.5 117 2.10 280 6.3 36 0.80 282 8.5 67 1.48 289 8.2 33 0.64 3047.2 161 3.91 320 6.3 43 0.83 322 15.2 168 3.27 340 6.0 109 2.64 361 10.471 1.73 379 9.5 78 1.75 402 39.9 135 3.14 022 16.2 83 1.69 040 7.8 671.43 048 6.5 43 0.94 055 11 55 1.04

TABLE II Tryptic peptides of the variable domains of the light chain ofK53/UBS54 and 02-237. Monoiso- Monoiso- First Last topic M_(W) (Da)topic M_(W) (Da) Peptide AA⁽¹⁾ AA K53/UBS54 02-237 L1 1 24 2580.31⁽²⁾2554.28⁽²⁾ L2 25 59 4039.02 4039.02 L3 60 66 700.35 700.35 L4 67 791302.61 1302.61 L5 80 82 374.23 374.23 L6 83 107 2810.29⁽²⁾ 2810.29⁽²⁾L7 108 111 487.30 487.30 L8 112 112 174.11 174.11 ⁽¹⁾AA, amino acid⁽²⁾One Cysteine residue alkylated

TABLE III Tryptic peptides of variable domains of heavy chains of K53,02-237 and UBS54. K53 02-237 UBS54 A B C D A B C D A B C D H1  1 121267.68 H1  1 12 1267.68 H1  1 12 1267.68 H2 13 19  685.41 H2 13 19 685.41

H3 20 23  492.24 H3 20 23  492.24 H3 20 23  492.24 H4 24 38 1693.81 H424 38 1693.81

H5 39 63 2783.28 H5 39 63 2783.28

H6 64 67  472.28 H6 64 67  472.28

H7 68 84 1906.87 H7 68 84 1906.87

H8 85 87  374.23 H8 85 87  374.23 — — — — H9 88 98 1319.55 H9 88 981319.55

— — — — Key: A: peptide B: first amino acid C: last amino acid D:monoisotopic M_(w) (Da) Remarks: 1) for H1, amino acid residue 1 is apyroglutamic acid 2) peptides H3 and H9 from K53 and 02-237, andpeptides H3 and H8 of UBS54 contain one alkylated cysteine residue 3)Unique peptides that can be used to confirm the presence of therespective IgGs are indicated in bold italics

TABLE IV MS/MS-data of CDR3 peptide (H11) of K53, obtained by collisioninduced dissociation of doubly charged m/z 1059.06. Ion m/z Y″₁ 147.12Y″₂ 248.18 Y″₃   335.21 ⁽¹⁾ Y″₄ 406.25 Y″₅ 507.30 Y″₆ 594.33 Y″₇ 693.40Y″₈ 794.46 Y″₉ 893.54 Y″₁₀ 1006.63  Y″₁₁ 1107.67  Y″₁₂ 1164.68  Y″₁₃1292.81  Y″₁₄ 1349.77  Y″₁₅ 1535.85  Y″₁₆ 1698.95  Y″₁₇ 1813.95  Y″₁₈1960.97  Y″₁₉  n.d.⁽²⁾ B₁ n.d. B₂ 157.10 B₃ 304.18 B₄ 419.22 B₅ 582.31B₆ 768.38 B₇ 825.39 B₈ 953.43 B₉ n.d. B₁₀ n.d. B₁₁ 1224.65  B₁₂ 1323.68 B₁₃ 1424.79  B₁₄ 1523.86  B₁₅ n.d. B₁₆ n.d. B₁₇ 1782.96  B₁₈ n.d. B₁₉n.d. ⁽¹⁾Underlined m/z-values are main peaks in the MS/MS-spectrum.⁽²⁾n.d. is not detected.

TABLE V MS/MS-data of CDR3 peptide (H11) of 02-237, obtained bycollision induced dissociation of doubly charged m/z 1030.02. Ion m/zY″₁ 147.12 Y″₂ 248.18 Y″₃ 335.20 Y″₄ 406.24 Y″₅ 493.30 Y″₆ 580.32 Y″₇679.40 Y″₈ 780.44 Y″₉ 879.53 Y″₁₀ 992.60 Y″₁₁ 1093.65  Y″₁₂ 1150.67 Y″₁₃ 1278.80  Y″₁₄ 1335.80  Y″₁₅ 1521.83  Y″₁₆ 1608.90  Y″₁₇ 1724.00 Y″₁₈ n.d. Y″₁₉ n.d. B₁ n.d. B₂ 189.09 B₃ n.d. B₄ 451.22 B₅ n.d. B₆ n.d.B₇ n.d. B₈ n.d. B₉ n.d. B₁₀ n.d. B₁₁ n.d. B₁₂ n.d. B₁₃ n.d. B₁₄ n.d. B₁₅n.d. B₁₆ n.d. B₁₇ n.d. B₁₈ n.d. B₁₉ n.d. ¹ Underlined m/z-values aremain peaks in the MS/MS-spectrum. ² n.d. is not detected.

TABLE VI MS/MS-data of CDR3 peptide (H9) of UBS54, obtained by collisioninduced dissociation of triply charged m/z 770.09. Ion m/z Y″₁ n.d. Y″₂248.17 Y″₃ 335.20 Y″₄ 406.25 Y″₅ 507.30 Y″₆ 594.33 Y″₇ 693.42 Y″₈ 794.45Y″₉ 893.53 Y″₁₀ 1006.64  Y″₁₁ 1107.67  Y″₁₂ 1164.68  Y″₁₃ n.d. Y″₁₄ n.d.Y″₁₅ n.d. Y″₁₆ n.d. Y″₁₇ n.d. Y″₁₈ n.d. Y″₁₉ n.d. Y″₂₀ n.d. B₁ n.d. B₂213.17 B₃ 360.16 B₄ 473.27 B₅ 610.32 B₆ 773.41 B₇ 959.48 B₈ 1016.50  B₉1144.57  B₁₀ 1201.59  B₁₁ 1302.68  B₁₂ 1415.72  B₁₃ 1514.78  B₁₄ n.d.B₁₅ n.d. B₁₆ n.d. B₁₇ n.d. B₁₈ n.d. B₁₉ n.d. B₂₀ n.d. ¹ Underlinedm/z-values are main peaks in the MS/MS-spectrum. ² n.d. is not detected.

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1. A method of producing a host cell, said method comprising:introducing into a host cell at least one nucleic acid sequence encodingan immunoglobulin light chain and nucleic acid sequences encoding atleast three different immunoglobulin heavy chains, wherein said at leastthree different immunoglobulin heavy chains are capable of pairing withsaid immunoglobulin light chain to form functional antigen bindingdomains of three or more non-identical antibodies.
 2. The methodaccording to claim 1, wherein said three or more non-identicalantibodies have differing specificities for the same target antigen. 3.The method according to claim 1, wherein said three or morenon-identical antibodies have differing affinities for the same targetepitope.
 4. The method according to claim 1, wherein said at least onenucleic acid sequence is present on a low copy number vector and saidhost cell produces 1 to 20 picograms per cell per day of said three ormore non-identical antibodies.
 5. The method according to claim 1,wherein said host cell comprises a human embryonic retina cell, andwherein said host cell has been immortalized or transformed byadenoviral E1 nucleic acid sequences.
 6. The method according to claim1, wherein said three or more non-identical antibodies are of differentisotypes.
 7. The method according to claim 6, wherein said differentisotypes comprise at least an IgG and an IgA.
 8. The method according toclaim 6, wherein said different isotypes comprise at least an IgG1 andan IgG3 antibody.
 9. The method according to claim 1, wherein said threeor more non-identical antibodies are independently selected from thegroup consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE andIgM.
 10. The method according to claim 1, wherein said at least threedifferent immunoglobulin heavy chains are of IgM isotype.
 11. The methodaccording to claim 1, wherein said at least three differentimmunoglobulin heavy chains are of an IgA isotype, and wherein at leasttwo of said three or more non-identical antibodies form a dimeric IgAantibody, said dimeric IgA antibody having non-identical binding sites.12. The method according to claim 1, wherein said three or morenon-identical antibodies bind to different epitopes of at least onetarget antigen.
 13. The method according to claim 1, wherein at leastone of said at least one nucleic acid sequence is stably expressed insaid host cell.
 14. The method according to claim 1, wherein said threeor more non-identical antibodies are produced by said host cell invitro.
 15. The method according to claim 1, wherein said at least threedifferent immunoglobulin heavy chains differ in at least one constantregion, said difference in at least one constant region reducingnon-functional antibody chain pairing.
 16. The method according to claim1, wherein said three or more non-identical antibodies comprise at leastone bispecific antibody.
 17. The method according to claim 1, whereinsaid three or more non-identical antibodies bind to different antigens.18. The method according to claim 1, wherein said three or morenon-identical antibodies bind synergistically to a target antigen. 19.The method according to claim 1, wherein a first antibody of the threeor more non-identical antibodies binds CD22, a second antibody of thethree or more non-identical antibodies binds CD72, and a third antibodyof the three or more non-identical antibodies binds HLA-DR.
 20. Themethod according to claim 1, wherein a first antibody of the three ormore non-identical antibodies binds an EP-CAM homotypic adhesionmolecule, and wherein a second antibody and a third antibody of thethree or more non-identical antibodies each bind CD46.
 21. The methodaccording to claim 1, wherein at least three of the three or morenon-identical antibodies bind to non-overlapping epitopes on Her-2. 22.The method according to claim 1, wherein the three or more non-identicalantibodies bind to targets selected from the group consisting of aHer-2/Neu receptor, a VEGFR1 receptor, a VEGFR2 receptor, a B-cellmarker, a T-cell marker, cytokines, interleukins, and cytokinereceptors.
 23. A method of producing a host cell, said methodcomprising: providing a host cell comprising a nucleic acid sequenceencoding an immunoglobulin light chain; introducing nucleic acidsequences encoding at least three different immunoglobulin heavy chainsinto said host cell, wherein said at least three differentimmunoglobulin heavy chains are capable of pairing with saidimmunoglobulin light chain to form functional antigen binding domains ofthe three or more non-identical antibodies.